PrimerDesign Catalogue/ Technical advice

Products for real-time PCR by Primerdesign

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Contents

3 Introduction4 genesig® Easy DNA/RNA Extraction Kit6 genesig® Easy Magnetic Rack8 genesig® Easy LysoBead Direct-to-PCR Extraction Kit10 Precision™ DNase Kits12 Precision™ Reverse Transcription Kits14 Precision™ Reverse Transcription Premix16 geNorm™ Reference Gene Selection Kits18 geNormPLUS Reference Gene Selection Kits20 qbase+ 22 Reference Gene Assays24 Custom Designed Real-Time PCR Assays for unusual species genes of interest26 Custom Designed Real-Time PCR Assays for any SNP, mutation or deletion in any species 27 About Our Custom Designed Real-Time PCR Assays28 Custom Designed Genotyping Assays 30 genesig® q16 and genesig Kits 32 Precision®PLUS qPCR Master Mix for human, mouse or rat genes of interest34 Precision®FAST qPCR Master Mix for unusual species genes of interest

36 Precision®PLUS OneStep RT-qPCR Master Mix38 Precision®ULTRA qPCR Master Mix40 Precision®ULTRA OneStep RT-qPCR Master Mix42 Precision®MULTIPLEX qPCR Master Mix44 Precision®MULTIPLEX OneStep RT-qPCR Master Mix46 Precision®CONTROL qPCR Master Mix48 Precision® HRM Master Mix50 oasig™Lyophilised 2X qPCR and oasig™ Lyophilised 2X OneStep RT-qPCR Master Mix52 BrightWhite Real-Time PCR Plasticware54 genesig® Easy Tips55 RNase/DNase Free Water 56 BioBank Control cDNA58 Real-Time PCR Internal Control Kit60 Genomic DNA Detection Kits61 Y Chromosome Detection Kits62 Molecular Diagnostics (MDx) Assay Development Services64 Technical Bulletins97 Appendix

Leaders in real-time

PCR assays

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Our core expertise is in the development of real-time PCR assays. We can produce real-time PCR assays for any gene of interest in any species. Our kits are available in a range of formats but, whatever your preference, you can be sure that every set of primers is designed by an expert, synthesised, and then fully validated on real biological cDNA in our laboratories to ensure that it is guaranteed to work to the highest standards on the day the kit arrives with you.

Uniquely, we will also provide all of the primer details for any custom designed assays that we produce for you to give you complete scientific integrity in your research. This makes our kits compliant with the The MIQE Guidelines: Minimum Information for Publication of Quantitative real-time PCR Experiments; Bustin S.A., et al 2009. Clinical Chemistry 55(4) 611–622

In addition to the World’s best custom designed primers, we can also provide everything else you need to perform real-time PCR to the highest standards whilst keeping your costs under control.

And of course, our dedicated, friendly team of experts are available to support you technically throughout your project to help you achieve the highest quality of data for your next publication or project.

Introduction

We founded Primerdesign in 2004 to share the expertise in real-time PCR and DNA chemistry found within the University of Southampton. Since then we have grown substantially, developing thousands of products, helping thousands of customers and working with customers in 100+ countries around the World.

DNA & RNA extraction from

virtually any sample

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genesig® Easy DNA/RNA Extraction Kit

Universal kit for isolation of DNA/RNA from food, water, clinical, veterinary and other samples types.

The genesig Easy DNA/RNA Extraction Kit allows DNA and RNA extraction from virtually any sample type using magnetic bead technology - it’s fast, and incredibly easy to perform.

The genesig Easy DNA/RNA extraction protocol begins with a simple lysis step where cells and tissue are lysed to release their nucleic acid. Then tiny magnetic particles are added to bind to DNA/RNA. When placed on to the genesig magnetic separator the particles are pulled to the side of the tube making it easy to remove the unwanted supernatant with a pipette. Then a series of simple wash steps are performed before the DNA/RNA is washed off the beads back in to solution, ready for analysis by real-time PCR.

Suitable sample types:

• Clinical and Veterinary - including whole blood, plasma, and serum; saliva and sputum; faeces and urine; tissues; and bacterial culture broth and swabs

• Food - including meat, fish, alcohol, milk, and cooked or processed food products

•Environmental - including plant*, soil, and water

*using genesig Easy DNA/RNA Plant Extraction Kit

Product features• Extracts DNA and RNA with high yields in 60 minutes•Works with huge range of sample types •Safe protocol with no phenolic chemicals• No centrifuge required - for use in the lab or in the field

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GENESIG EASY DNA/RNA EXTRACTION KITS

CATALOGUE NO. PRODUCT DESCRIPTION KIT SIzE

genesigEASY-EK genesig Easy DNA/RNA Extraction Kit 50 extractions

genesigEASY-EK-PLANT genesig Easy DNA/RNA Plant Extraction Kit 50 extractions

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genesig® Easy Magnetic Rack

The genesig Easy Magnetic Rack for use with the genesig Easy DNA/RNA Extraction Kit enables simple nucleic acid extraction from up to 16 different samples at once. Powerful magnets and angled sides create very tight magnetic bead clumps. This makes it simple to aspirate from the tube with a pipette without the risk of disturbing your magnetic beads.

The detachable tube holder makes it convenient to handle multiple samples simultaneously. When used in combination with the genesig Easy DNA/RNA Extraction Kit, nucleic acid extraction from virtually any sample is made easy.

Product features•For use with genesig Easy DNA/RNA Extraction Kit• Powerful magnets for tight magnetic bead clumps and easy

aspiration• Extraction from up to 16 different samples at once

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GENESIG EASY MAGNETIC RACK


genesigEASY-MR genesig Easy Magnetic Rack for DNA/RNA extraction 1 rack for 16 samples

Extraction from up to 16

different samples at once

The genesig Easy LysoBead Direct-to-PCR Extraction Kit was used to extract DNA from a sample containing Microsporum canis, which is known for being a more problematic sample type.

Amplification of Microsporum canis extracted DNA was conducted on the Bio-Rad CFX instru-ment using the Primerdesign Microsporum canis assay together with PrecisionPLUS qPCR Master Mix. A generic standard cycling protocol was applied to produce the amplification shown.

The genesig Easy LysoBead Direct-to-PCR Extraction Kit produced high quality and reliable DNA extraction from Microsporum canis.

Amplification curves: FAM channelLight-blue - genesig® Easy LysoBead Direct-to-PCR Extraction Kit Blue - Negative extraction control

Cycles

Amplification

0

0

1000

2000

3000

4000

5000

10 20 30 40 50

RFU

(10^

3)

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genesig® Easy LysoBead Direct-to-PCR Extraction KitThe new genesig Easy LysoBead Direct-to-PCR Extraction Kit allows rapid isolation of highly purified DNA suitable for any down-stream amplification reaction such as qPCR. Reliable DNA extraction can be obtained from the smallest amounts of source material (5 to 25mg or 100µl).

The protocol incorporates disruption bead technology into a PCR-ready lysis formulation, allowing for a single tube preparation of DNA samples for molecular testing in under 10 minutes.

This is a significant reduction in the time it takes to extract DNA from microbes with highly developed cell walls, such as gram positive bacteria, fungal and mycobacteria samples, where the lysis stage was typically augmented with either a prolonged overnight incubation, or with mechanical disruption steps.

Suitable sample types:

• Clinical and Veterinary - including swabs and urine•Food - including meat, fish, and milk; excluding processed food

products Suitable for DNA extraction from difficult to lyse pathogens:

• Bacteria (Gram +ve/-ve) - including Chlamydia, Clostridium and Streptococcus

• Virus - including HBV, HPV, HSV, and fish viruses• Fungi, Yeast, Mycoplasma, Mycobacterium, and Protozoa

Product features• Fast DNA extraction in under 10 minutes• User friendly 5 step protocol in one tube• Works with huge range of difficult sample types• Cost effective with no expensive equipment required

Amplification graph showing reliable DNA extraction from Microsporum canis using genesig® Easy LysoBead Direct-to-PCR Extraction Kit

User friendly 5-step protocol

in one tube

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GENESIG EASY LYSOBEAD DIRECT-TO-PCR EXTRACTION KIT


genesigEASY-LysoBead genesig Easy LysoBead Direct-to-PCR Extraction Kit 50 extractions

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Precision™ DNase Kits

Precision DNase is a novel product designed specifically for removing gDNA contamination from RNA prior to reverse transcription/real-time PCR. The Precision DNase enzyme has an exceptional level of specificity towards dsDNA and no activity against RNA. In addition the kit’s novel deactivation mechanism results in pure, un-degraded RNA.

Unique ‘stop’ method protects RNA; our enzyme is inactivated by a short incubation at just 55°C. Heat inactivation above 65°C, as common for other enzymes, can cause magnesium mediated degradation of RNA. This simple inactivation method ensures that RNA is both protected from degradation and prevents the need for additional reagents to stop the reaction that may affect sample quality.

Other DNase kits available are unsuitable for preparing RNA for real-time PCR.

•Many use an aggressive heat inactivation (>65°C) which can result in magnesium mediated RNA degradation

•Some use a chemical stop solution containing EDTA (a known disrupter of PCR)

•Whilst other kits offer a slurry of beads that capture the enzyme and allow centrifugal removal. This final approach makes recovering all of the RNA sample impossible

Our Precision DNase kit is unique in having a heat labile enzyme. This means that enzyme activity is entirely destroyed by an ‘RNA safe’ 5 minute incubation at 65°C.

Product features•Precise and efficient gDNA removal in 15 minutes •No damage to RNA •No addition of EDTA (PCR inhibitor) •100% recovery of all RNA

This product has been designed and manufactured in collaboration with Articzymes AS

Precise and efficient gDNA

removal in 15 minutes

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PRECISION DNASE KIT


DNase-50 Precision DNase kit 50 reactions

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High efficiency reverse transcription (RT) ensures the ability to detect rare transcripts and obtain the strongest possible real-time PCR signals. As a complete solution, the Primerdesign Reverse Transcription kit contains all of the reagents necessary. Both Oligo(dT) and random nonamers are included for complete flexibility. For advice on the most appropriate priming strategy contact our team of experts on [email protected]

Our reverse transcriptase are especially modified to work at high temperatures. Higher reaction temperatures reduce the RNA secondary structure (tangling), allow the production of longer transcripts and make for faster reactions. The enzyme also has an increased affinity for primer-template complexes which allow for efficient transcription of very low amounts of RNA.

Product features•Cost saving•Precise, reproducible results•Ultra fast protocol •Flexible kit design - Oligo(dT), random nonamer

Kit contents•High quality nanoScript2 RT enzyme•10 x RT buffer•DTT•Oligo(dT) primers•Random nonamer primers•dNTPs•RNase/DNase-free water

Precision™ Reverse Transcription Kits

Precise, reproducible

results

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PRECISION REVERSE TRANSCRIPTION KITS


RT-nanoScript2 nanoScript2 Reverse Transcription kit suitable for 2 nanograms to 2 micrograms of RNA template

50 reactions

The ultimate convenience

for cDNA synthesis

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Our all-in-one RT products provide the ultimate convenience for cDNA synthesis.

The pre-optimised mix allows for rapid reverse transcription in a single closed tube reaction. Our proprietary formulation contains an optimised blend of oligo(dT) and random nonamer primers, buffer, dNTPs, and RT enzyme. Simply add RNA to the mix, incubate at 55°C for 20 minutes and your perfect reverse transcription reaction is complete.

Product features•Ultimate convenience •Reduced contamination risks •Reduced handling errors•Ultra fast protocol

Precision™ Reverse Transcription Premix

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REVERSE TRANSCRIPTION PREMIX


RT-premix2 All-in-one Reverse Transcription mix. Single tube for manual dispensing. 50 reactions

RT-noRT-ctrl No RT control aliquot (complete mix lacking the RT enzyme) 50 reactions

MIQE guidelines compliant

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For accurate gene quantification, it is essential to normalise real-time PCR data to a fixed reference; one that is not affected by your experimental conditions. There is increasing evidence that normalising to a single, randomly selected reference (housekeeping) gene introduces large and variable errors into the analysis. The geNorm kit is a system for selecting the best candidate reference genes for a given experimental scenario.

>12,000 papers have already cited the geNorm method.

To perform geNorm analysis, the user measures the expression in their samples of the panel of candidate reference genes in the kit. The geNorm software qbase+ provided with the kit ranks the reference genes in order of stability. The output graphs identify the best reference genes for that particular experiment and also the optimum number that should be included for accurate normalisation.

Our classic geNorm kit contains the most commonly used reference genes in the literature plus additional genes from a wide variety of cellular processes. A full list of the genes in each kit is available on our website.

Product features•Quick simple protocol• Identifies the best reference genes for any given

experimental model• Improves accuracy, sensitivity and reproducibility of

subsequent experiments•Qualify for 50% off future reference gene assay orders*

Kit contents•Lyophilised primers (and probe) for 6 or 12 reference genes (200

reactions each)•RNase/DNase free water•Access to the latest geNorm software in qbase+

•50% off discount code for future reference gene assay orders*

In addition to human, mouse and rat we have geNorm kits for many other research species including E.Coli, Drosophila, Horse, Xenopus, Sheep, zebra Fish, Chicken, Pig, Rabbit.

See our website for the latest complete list. If you would like to use geNorm for a species that we do not currently support then please contact us as we can develop kits on demand.

In collaboration with

geNorm™ Reference Gene Selection Kits

*T&C’s apply

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GENORM™ REFERENCE GENE SELECTION KITS

HUMAN


ge-SY-6-hu ge-SY-12-hu

6 gene human geNorm™ kit for use with SYBR® Green12 gene human geNorm™ kit for use with SYBR® Green

6 x 200 reactions 12 x 200 reactions

ge-DD-6-hu ge-DD-12-hu

6 gene human geNorm™ kit with Double-Dye hydrolysis probe12 gene human geNorm™ kit with Double-Dye hydrolysis probe


MOUSE


ge-SY-6-mo ge-SY-12-mo

6 gene mouse geNorm™ kit for use with SYBR® Green12 gene mouse geNorm™ kit for use with SYBR® Green


ge-DD-6-mo ge-DD-12-mo

6 gene mouse geNorm™ kit with Double-Dye hydrolysis probe12 gene mouse geNorm™ kit with Double-Dye hydrolysis probe


RAT


ge-SY-6-rage-SY-12-ra

6 gene rat geNorm™ kit for use with SYBR® Green12 gene rat geNorm™ kit for use with SYBR® Green


ge-DD-6-rage-DD-12-ra

6 gene rat geNorm™ kit with Double-Dye hydrolysis probe12 gene rat geNorm™ kit with Double-Dye hydrolysis probe

6 x 200 reactions12 x 200 reactions

Genome Biology, 2002, ‘Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes’ Vandesompele et al

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What is different about a geNormPLUS kit? In collaboration with Nebion (www.nebion.com), using their Genevestigator® technology, we screened data from >30,000 microarray experiments to identify genes that express inherent levels of stability across different tissues, alternative experimental treatments, different disease states and many other conditions. The result is a list of genes that are inherently stably expressed (the lists includes a selection of classically used reference (housekeeping) genes for comparison too). geNorm analysis with these genes therefore enables selection of the “best of the best”.

A complete list of the genes included can be found on our website.

Product features•Scientifically selected, inherently stable genes•Quick simple protocol• Identifies the best reference genes for any

given experimental model• Improves accuracy, sensitivity and reproducibility of

subsequent experiments•Qualify for 50% off future reference gene assay orders*


Kit contents•Lyophilised primers for 6 or 12 reference genes

(200 reactions each)•RNase/DNase free water•Access to the latest geNorm software in qbase+ •50% off discount code for future

reference gene assay orders*

geNormPLUS Reference Gene Selection Kits

*T&C’s apply

Scientifically selected, inherently

stable genes

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GENORMPLUS REFERENCE GENE SELECTION KITS

HUMAN


gePLUS-SY-6-hugePLUS -SY-12-hu

6 gene human geNormPLUS kit for use with SYBR® Green12 gene human geNormPLUS kit for use with SYBR® Green


MOUSE


gePLUS -SY-6-mogePLUS -SY-12-mo

6 gene mouse geNormPLUS kit for use with SYBR® Green12 gene mouse geNormPLUS kit for use with SYBR® Green


RAT


gePLUS -SY-6-ragePLUS -SY-12-ra

6 gene rat geNormPLUS kit for use with SYBR® Green12 gene rat geNormPLUS kit for use with SYBR® Green


The most powerful and user-friendly

software in the marketplace

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Primerdesign is delighted to have been chosen by Biogazelle to partner them in marketing their World famous qPCR analysis software qbase+. The software is recognised as the most powerful and user-friendly product in the marketplace. The software will completely automate analysis of your qPCR data whilst being flexible enough to produce the graphs and statistics that you need to tell your story of choice with your data.

Product features•Easy to use•Time saving•Flexible•Analysis wizard •Effortless scaling for larger experiments•Complete automation of statistics and graphing •Based on peer reviewed quantification models•MIQE and RDML ompliant


qbase+ the World’s best real-time PCR analysis software

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QBASE+

CATALOGUE NO. PRODUCT DESCRIPTION LICENSES AVAILABLE

qbase-acc-B qbase+ licences for academic users Premium 3 yearsPremium 1 yearPremium 2 monthsBasic 1 yearBasic 2 months

qbase-com-B qbase+ licences for commercial users Premium 3 yearsPremium 1 yearPremium 2 months Basic 1 yearBasic 2 months

For group licenses please enquire.

50% discount

available!*

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To compliment your custom gene of interest assay, Primerdesign has a wide range of reference gene assays which can be used as a normalising signal.

If you do not know the best reference genes for your experiment, you can use our geNorm kit to enable selection of the most stable genes in your individual experiment (see page 16). If you have used our geNorm kit previously, you qualify for a 50% discount on the purchase of reference (housekeeping) gene assays for future use*.

Please see Appendix on page 97 for a list of our available reference gene assays, or our website.

Reference gene assays for other speciesWe also hold reference gene assays for many other species including:

•Arabidopsis•C.elegans•Xenopus•Sheep•zebra Fish•Red Fire Ant•Pig•Rabbit

The list is constantly expanding so contact us for an up to date perspective.

If we do not already have the reference gene kits that you require then you can order them as custom gene of interest assays (see page 24).

Reference Gene Assays

*T&C’s apply

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REFERENCE GENE ASSAYS

HUMAN


HK-SY-hu-300 Pre-validated primers and hydrolysis probe (e.g. TaqMan®) for your choice of human reference gene

300 reactions

HK-DD-hu-600 Pre-validated primers for your choice of human reference gene for use with SYBR® Green

600 reactions

MOUSE


HK-SY-mo-300 Pre-validated primers and hydrolysis probe (e.g. TaqMan®) for your choice of mouse reference gene

300 reactions

HK-DD-mo-600 Pre-validated primers for your choice of mouse reference gene for use with SYBR® Green

600 reactions

RAT


HK-SY-ra-300 Pre-validated primers and hydrolysis probe (e.g. TaqMan®) for your choice of rat reference gene

300 reactions

HK-DD-ra-600 Pre-validated primers for your choice of rat reference gene for use with SYBR® Green

600 reactions

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Primerdesign specialise in the custom design and validation of real-time PCR primer assays. Kits are provided ready optimised and are guaranteed to work to the highest standards. Simply supply the name and species or accession number of your target gene of interest and our team of professional molecular biologists will design the best possible real-time PCR primers for that unique sequence.

Once synthesised our quality control team validate the performance of the primers on relevant biologically derived cDNA from a range of cell lines and tissue samples. When our expert team are satisfied with the performance of the kit, the primers (and probe) are supplied as a ready optimised mix and are guaranteed to give you high quality data without any further validation.

We are flexible; if you have specific design requirements we can incorporate these into your kit. e.g. splice variant detection, intron spanning amplicons etc. Indeed if you have any custom requirements, please enquire (surcharges may apply).

Product features•Complete primer details supplied with kit• Individual, expert designs•Guaranteed priming specificity•Guaranteed priming efficiency >90% •Fully optimised validated on relevant cDNA •MIQE guideline compliant

Kit contents•Lyophilised primers (and probe)•RNase/DNase free water•Primer data sheet

Custom Designed Real-Time PCR Assaysfor human, mouse or rat genes of interest

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CUSTOM DESIGNED REAL-TIME PCR ASSAYS FOR HUMAN, MOUSE OR RAT GENES OF INTEREST

HUMAN


SY-hu-600SY-hu-900SY-hu-1200

Custom designed primers for use with SYBR® Green for any human gene 600 reactions900 reactions1200 reactions

DD-hu-300DD-hu-600DD-hu-900

Custom designed primers with hydrolysis probe (e.g. TaqMan®) for any human gene

300 reactions600 reactions900 reactions

MOUSE


SY-mo-600SY-mo-900SY-mo-1200

Custom designed primers for use with SYBR® Green for any mouse gene 600 reactions900 reactions1200 reactions

DD-mo-300DD-mo-600DD-mo-900

Custom designed primers with hydrolysis probe (e.g. TaqMan®) for any mouse gene


RAT


SY-ra-600SY-ra-900SY-ra-1200

Custom designed primers for use with SYBR® Green for any rat gene 600 reactions900 reactions1200 reactions

DD-ra-300DD-ra-600DD-ra-900

Custom designed primers with hydrolysis probe (e.g. TaqMan®) for any rat gene 300 reactions600 reactions900 reactions

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Not everyone works on human, rat or mouse. Primerdesign provide a unique service in the design and validation of real-time PCR primer assays for any gene in any species. Kits are provided ready optimised and guaranteed to work to the highest standards. Simply supply the name, accession number or raw DNA sequence of your target gene. Our team of professional molecular biologists will design the best possible real-time PCR primers for that unique sequence.

Once synthesised, our quality control team test the performance of the primers on a synthetic DNA template. Alternatively we can test the primers on user supplied cDNA (please contact us if you require this service). Only then are our kits packaged and shipped ready for use.

We are flexible; if you have specific design requirements we can incorporate these into your kit. e.g. splice variant detection, intron spanning amplicons etc. Indeed if you have any custom requirements, please enquire (surcharges may apply).

Product features•Complete primer details supplied with kit• Individual, expert designs•Guaranteed priming specificity•Guaranteed priming efficiency >90% •Fully wet test validated

Kit contents•Lyophilised primers (and probe)•RNase/DNase free water•Primer data sheet

Custom Designed Real-Time PCR Assays for unusual species genes of interest

CUSTOM DESIGNED REAL-TIME PCR ASSAYS FOR UNUSUAL SPECIES GENES OF INTEREST


SY-any-600SY-any-900SY-any-1200

Custom designed primers for use with SYBR® Green for non-standard species 600 reactions900 reactions1200 reactions

DD-any-300DD-any-600DD-any-900

Custom designed primers with hydrolysis probe (e.g. TaqMan®) for non-standard species


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About Our Custom Designed Real-Time PCR Assays

Primer only (for use with SYBR® Green)Primer only kits are the most cost effective method for real-time PCR detection. Our design approach ensures high specificity binding and minimal PCR artefacts, such as primer dimers, which can reduce the effectiveness of this approach. ‘Primer only’ kits work with standard two step cycling conditions and work well with all high quality commercially available master mixes. However, we recommend the use of Primerdesign 2X PrecisionPLUS or PrecisionFAST (see pages 32 and 34).

Double-Dye probes (TaqMan® style hydrolysis probes)Hydrolysis probes (TaqMan®, Double-Dye) are the current industry standard* for real-time PCR detection. Assays are available in this format to support researchers who require this type of probe to compliment their existing approach to real-time PCR, and come with FAM dye as standard; if you have other dye requirements, please enquire (surcharges may apply). Our Double-Dye probes work with standard two step cycling conditions and work well with all high quality commercially available master mixes. However, we recommend the use of Primerdesign 2X PrecisionPLUS or PrecisionFAST (see pages 32 and 34).

*standard probe when probe based real-time PCR detection needs to be utilised.

Our expertise is in the rapid design and validation of real-time PCR detection kits for research purposes. We have the flexibility and expertise to offer real-time PCR solutions in any format. As standard, we offer two types of kit, primer only and hydrolysis probe. If you require something else then please enquire.

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Primerdesign specialise in designing the World’s best real-time PCR assays for genotyping. Assays are individually designed by an expert and then wet tested in our laboratories to ensure ideal performance.

They are supplied with complete disclosure of the primer sequences so that you are in complete control of your experiment.

Product features•Lyophilised primer/probe assay• Individual expert design•Expert validation process•Guaranteed data quality•Complete primer details supplied

Kit contents•Multiplex primer and probe set for SNP of interest•Positive control gDNA•RNase/DNase free water•Primer data sheets

Custom Designed Genotyping Assays for any SNP, mutation or deletion in any species

Lyophilised primer/ probe assay

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CUSTOM DESIGNED GENOTYPING ASSAYS FOR ANY SNP, MUTATION OR DELETION IN ANY SPECIES

GT-hu-750GT-hu-3000GT-hu-6000

Custom genotyping real-time PCR assays for any human target 750 reactions3000 reactions6000 reactions

GT-mo-750GT-mo-3000GT-mo-6000

Custom genotyping real-time PCR assays for any mouse target 750 reactions3000 reactions6000 reactions

GT-ra-750GT-ra-3000GT-ra-6000

Custom genotyping real-time PCR assays for any rat target 750 reactions3000 reactions6000 reactions

GT-any-750GT-any-3000GT-any-6000

Custom genotyping real-time PCR assays for any other species target 750 reactions3000 reactions6000 reactions

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Highly affordable qPCR instrumentThe genesig q16 is priced to make it affordable for any business to access the power of real-time PCR. Perfect for a whole new world of molecular testing customers.

Automated analysisThe genesig q16 makes real-time PCR easy for the first time. The genesig Easy Kit format is designed specifically for the q16 and simplifies everything during test set-up. The q16 software experience is fast and intuitive, whilst analysis of the data is completely automated meaning it can be used by an expert or a complete novice with equal, powerful results.

Groundbreaking technologyAs well as being remarkably beautiful, the genesig q16 is technologically powerful. It has no moving parts, making it very robust. It operates in silence and weighs less than 2kg. It can operate with a PC or Mac but can also operate in stand alone mode with the test data being downloaded on to a USB drive for analysis later.

A complete solutionThe genesig q16 can be used with regular lab equipment and DNA extraction systems. But if you don’t have a lab, the genesig Easy Lab-in-a-box and the genesig Easy DNA/RNA extraction kit will provide all that you need to complete your molecular testing set-up.

More than 600 genesig® kits• Exceptional value for money• Lyophilised for convenient shipping• Rapid detection of all clinically relevant subtypes• Positive copy number standard curve for quantification• Highly specific detection profile• High priming efficiency• Broad dynamic detection range (>6 logs)• Sensitive to <100 copies of target• Accurate controls to confirm findings

genesig is our range of molecular testing kits for real-time PCR. The huge menu has been developed by our expert team over the last 12 years and are used by high profile customers all over the World.

Kits contain copy number positive controls to allow for precise copy number determination, an internal extraction control to give detailed insight in to the success of your extraction process.

New genesig kits on demand: If your target of interest is missing from our huge menu we can develop a new test for you on demand in just 6 weeks.

Human pathogen detection kits: HCV, HBV, HIV, MRSA, HPV, CMV, Chlamydia, Dengue and many more.

Human pathogen multiplex detection kits: Dengue, Zika and Chikungunya Virus, Dengue subtyping, High Risk HPV.

Veterinary diagnostic kits: Bovine TB, Foot and Mouth disease, Blue Tongue, Taylorella equigenitalis, CDV, FIV and many more.

Food, water and agricultural testing kits: Listeria, Salmonella, Legionella, Horse speciation, Pork speciation and many more.

genesig® q16 and genesig Kits an extraordinary qPCR instrument and kits for real-time PCR

The World’s Most Affordable

qPCR Machine

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Comparison of Precision®PLUS qPCR Master Mix vs. other commercially available standard cycling qPCR master mixes

Amplification of the rat Mmp9 gene from rat cDNA. Testing was conducted on the Bio-Rad CFX instrument using the Primerdesign Mmp9 rat gene assay. A generic standard cycling protocol, with an extended enzyme activation step, was applied to enable parallel testing.

PrecisionPLUS qPCR Master Mix (in dark blue) is compared to 5 competitor master mixes in light blue (listed in order of trace appearance).

PrecisionPLUS qPCR Master Mix produced highly efficient, consistent and reliable detection in comparison to all of the competitor master mixes.

Cycles

Amplification

00

1000

2000

3000

4000

5000

6000

7000

10 20 30 40 50

RFU

(10^

3)

Amplification curves: FAM channel

PrecisionPLUS = 30.47Competitor B = 30.41Competitor R = 31.19Competitor T = 32.23Competitor L = 32.29Competitor Q = 34.99

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High quality, robust 2X qPCR master mix at an exceptionally low cost. The core components are a hot start Taq polymerase enzyme with a magnesium chloride based buffer. Stabilisers and preservatives ensure that multiple freeze-thaw cycles do not affect the performance. It contains a high fidelity enzyme mix which will provide for accurate quantitative measurements for qPCR.

Primerdesign real-time PCR reagents are manufactured to the highest standards. Free samples are available for validation purposes, please enquire.

Product features•Huge cost saving•Precise reproducible results•High efficiency enzyme - better data• Robust •Available for all machines (see website for complete list)•SYBR® Green dye added free of charge• Inert blue loading dye added free of charge

All our PrecisionPLUS master mixes are available with or without SYBR® Green premixed at no extra charge. Simply state your preference when ordering or add “-SY” to the catalogue number for clarity.

Precision®PLUS qPCR Master Mix

Huge cost

savings!

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PRECISIONPLUS QPCR MASTER MIX


PPLUS-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix 1ml; 2ml; 5ml; 10ml; 20ml

PPLUS-R-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix with ROX* 1ml; 2ml; 5ml; 10ml; 20ml

PPLUS-LR-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix with low ROX** 1ml; 2ml; 5ml; 10ml; 20ml

PPLUS-iC-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix for the Bio-Rad 1ml; 2ml; 5ml; 10ml; 20ml

PPLUS-CL-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix for the Roche LightCycler 1.0-2.0 1ml; 2ml; 5ml; 10ml; 20ml

PPLUS-MX-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS qPCR Master Mix for Stratagene MX machines 1ml; 2ml; 5ml; 10ml; 20ml

*Recommended for ABI 7000, 7300, 7700, 7900, StepOne and StepOnePlus - If your machine is not specified please enquire **Recommended for ABI 7500, ViiA7 and QuantStudio - if your machine is not specified please enquire.

Comparison of Precision®FAST qPCR Master Mix vs. other commercially available fast cycling qPCR master mixes

Amplification of the rat Mmp9 gene from rat cDNA. Testing was conducted on the Bio-Rad CFX instrument using the Primerdesign Mmp9 rat gene assay. A generic fast cycling protocol, with an extended enzyme activation step, was applied to enable parallel testing.

PrecisionFAST qPCR Master Mix (in dark blue) is compared to 4 competitor fast master mixes in light blue (listed in order of trace appearance).

PrecisionFAST qPCR Master Mix produced rapid detection whilst maintaining the highest standards of efficiency, consistency and reliability in comparison to the competitor fast master mixes.

Cycles

Amplification

00

5

10

15

20

25

10 20 30 40 50

RFU

(10^

3)


PrecisionFAST = 29.79 CqCompetitor K = 30.06 CqCompetitor T = 30.53 CqCompetitor Q = 32.86 CqCompetitor R = 47.93 Cq

34

Precision®FAST qPCR Master Mix

PrecisionFAST qPCR master mix is an ultra-fast, cost-saving mix for qPCR. The 2X mix is designed for rapid cycling protocols that can dramatically shorten run times. The Taq polymerase has been mutated at the active site and has higher affinity for DNA and faster processing. The buffer has been designed for optimum sensitivity and also to reduce primer dimers which are a common artefact of fast processing enzymes.

Product features•Ultra-fast enzyme•qPCR data in 40 minutes•Available for all machines (see website for complete list)•Minimises primer dimer formation•SYBR® Green dye added free of charge• Inert blue loading dye added free of charge

All our PrecisionFAST master mixes are available with or without SYBR® Green premixed at no extra charge. Simply state your preference when ordering or add “-SY” to the catalogue number for clarity.

qPCR data in

~40 minutes

35

PRECISIONFAST QPCR MASTER MIX


PFAST-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix 1ml; 2ml; 5ml; 10ml; 20ml

PFAST-R-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix with ROX* 1ml; 2ml; 5ml; 10ml; 20ml

PFAST-LR-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix with low ROX** 1ml; 2ml; 5ml; 10ml; 20ml

PFAST-iC-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix for Bio-Rad machines 1ml; 2ml; 5ml; 10ml; 20ml

PFAST-CL-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix for the Roche LightCycler 1.0-2.0 1ml; 2ml; 5ml; 10ml; 20ml

PFAST-MX-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionFAST qPCR Master Mix for Stratgene MX machines 1ml; 2ml; 5ml; 10ml; 20ml


36

Our PrecisionPLUS OneStep RT-qPCR Master Mix contains all of the required components for a perfect one step real-time PCR analysis in a single reaction mix. The operator simply adds RNA and a primer/probe mix for a complete, closed tube, one step RNA to Cq reaction. NanoScript2 is a modified thermostable MMLV enzyme with an optimum working temperature of 55°C and a higher affinity for primer duplex initiation sites.

This increases the rate of processing and the sensitivity of detection. The result is an RT step that is completed in less than 10 minutes and is sensitive in a linear range across the whole biological scope from very high (>1 x 109 copies) to very low (<10 copies) RNA copy numbers. An RNase inhibitor is included to protect the sample from possible degradation.

Product features•Convenient, one step, closed tube reaction•Rapid protocol - includes 10 minute RT step• Ideal for RNA pathogen detection•Reduced handling/set up time•Reduced risk of contamination•SYBR® Green dye added free of charge• Inert blue loading dye added free of charge

Precision®PLUS OneStep RT-qPCR Master Mix

Comparison of Precision®PLUS OneStep RT-qPCR Master Mix vs. other commercially available one step RT-qPCR master mixes

Amplification of the rat Mmp9 gene from rat RNA. Testing was conducted on the Bio-Rad CFX instrument using the Primerdesign Mmp9 rat gene assay. A generic one-step protocol, with an extended enzyme activation step, was applied to enable parallel testing. The reveres transcrip-tion stage was applied as a gradient to ensure all master mixes were utilised appropriately.

PrecisionPLUS OneStep RT-qPCR Master Mix (in dark blue) is compared to 6 competitor master mixes in light blue (listed in order of trace appearance).

PrecisionPLUS OneStep RT-qPCR Master Mix produced highly efficient, consistent and reliable detection in comparison to all of the competitor master mixes.

Cycles

Amplification

0

0

1000

2000

3000

4000

5000

6000

7000

10 20 30 40

RFU

(10^

3)


PrecisionPLUS OneStep = 28.34Competitor B = 28.57 Competitor T = 28.84 Competitor L = 29.05Competitor K = 29.16 Competitor Q = 38.40Competitor R = 39.99

37

PRECISIONPLUS ONESTEP RT-QPCR MASTER MIX


OSPLUS-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix 1ml; 2ml; 5ml; 10ml; 20ml

OSPLUS-R-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix with ROX* 1ml; 2ml; 5ml; 10ml; 20ml

OSPLUS-LR-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix with low ROX** 1ml; 2ml; 5ml; 10ml; 20ml

OSPLUS-iC-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix for the Bio-Rad iCycler 1ml; 2ml; 5ml; 10ml; 20ml

OSPLUS-CL-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix for the Roche LightCycler 1.0-2.0 1ml; 2ml; 5ml; 10ml; 20ml

OSPLUS-MX-1ml; 2ml; 5ml; 10ml; 20ml

PrecisionPLUS OneStep RT-qPCR Master Mix for Strategene MX machines 1ml; 2ml; 5ml; 10ml; 20ml


38

PrecisionULTRA qPCR master mix can help when you are dealing with samples with very low copy numbers, and is optimised for applications such as ccfDNA qPCR analysis. This 2X master mix contains a unique formulation to increase the sensitivity of your testing.

The core components are a hot start Taq polymerase enzyme with a unique ammonium sulphate based buffer optimised to reduce interference of non-specific binding. Our unique formulation gives a superior quality of data with a bigger signal to noise ratio, brighter, steeper and more precise amplification plots. Stabilisers and preservatives ensure that multiple freeze-thaw cycles do not affect the performance giving you reliable, reproducible results.

Product features•Optimised for low copy number samples•Precise reproducible results across wide range of template

amounts•Optimised to run on fast and standard cycling protocols•Exceptional value for money•SYBR® Green dye added free of charge• Inert blue loading dye added free of charge

Precision®ULTRA qPCR Master Mix

Amplification graph demonstrating the improved low copy amplification profile introduced by Precision®ULTRA qPCR Master Mix

During the course of the performance investigation, PrecisionULTRA qPCR Master Mix produced an improvement in the amplification profile of detected low copy number samples.

The data presented here reflects the amplification curves produced by both PrecisionULTRA qPCR Master Mix and a standard master mix in the detection of samples diluted down to 1 copy (per reaction).

The amplification curves produced by PrecisionULTRA qPCR Master Mix represents a higher end-point fluorescence and a stronger sigmoidal shape/function, indicating a more efficient PCR.

1 copy detection with the genesig WSSV Standard Kit

Blue (FAM) = PrecisionULTRA qPCR Master Mix (19/30 replicates detected)Light blue (FAM) = Standard Master Mix (18/30 replicates detected)

Cycles

Amplification

0

0

5

10

15

20

25

10 20 30 40 50

RFU

(10^

3)

Superior quality of data

with bigger signal to noise ration

39

PRECISIONULTRA QPCR MASTER MIX


PULTRA-10ML PrecisionULTRA qPCR Master Mix 10ml: 10 x 1.0ml

PULTRA-R-10ML PrecisionULTRA qPCR Master Mix with ROX* 10ml: 10 x 1.0ml

PULTRA-LR-10ML PrecisionULTRA qPCR Master Mix with low ROX** 10ml: 10 x 1.0ml

PULTRA-iC-10ML PrecisionULTRA qPCR Master Mix for Bio-Rad machines 10ml: 10 x 1.0ml

PULTRA-CL-10ML PrecisionULTRA qPCR Master Mix for Roche LightCycler 1.0-2.0 10ml: 10 x 1.0ml

PULTRA-MX-10ML PrecisionULTRA qPCR Master Mix for Stratagene MX machines 10ml: 10 x 1.0ml


Supreme sensitivity at the limit of detection

40

PrecisionULTRA OneStep RT-qPCR Master Mix can help when you are dealing with samples with very low copy numbers. This 2X master mix contains a unique formulation to increase the sensitivity of testing RNA or DNA targets simultaneously.

The core components are a hot start Taq polymerase and a Reverse Transcriptase enzyme, including a unique ammonium sulphate based buffer optimised to reduce interference of non-specific binding. Our unique formulation gives a superior quality of data with a bigger signal to noise ratio, brighter, steeper and more precise amplification plots. Stabilisers and preservatives ensure that multiple freeze-thaw cycles do not affect the performance giving you reliable, reproducible results.

Product features•Optimised for low copy number samples•Precise reproducible results across wide range of template

amounts•Suitable for both RNA and DNA targets•Optimised to run on fast and standard cycling protocols•Exceptional value for money•SYBR® Green dye added free of charge• Inert blue loading dye added free of charge

Precision®ULTRA OneStep RT-qPCR Master Mix

Amplification graph demonstrating the improved low copy amplification profile introduced by Precision®ULTRA OneStep RT-qPCR Master Mix

During the course of the performance investigation, PrecisionULTRA OneStep RT-qPCR Master Mix produced an improvement in the amplification profile of detected low copy number samples.

The data presented here reflects the amplification curves produced by both PrecisionULTRA OneStep RT-qPCR Master Mix and a standard one step master mix in the detection of samples diluted down to 1 copy (per reaction).

The amplification curves produced by PrecisionULTRA OneStep RT-qPCR Master Mix represents a higher end-point fluorescence and a stronger sigmoidal shape/function, indicating a more efficient PCR.

Cycles

Amplification

00

2

4

6

8

10

10 20 30 40 50

RFU

(10^

3)

1 copy detection with the genesig Human Immunodeficiency Virus Type 1 Standard Kit

Blue (FAM) = PrecisionULTRA OneStep RT-qPCR Master Mix (17/30 replicates detected)Light blue (FAM) = Standard OneStep Master Mix (16/30 replicates detected)

41

PRECISIONULTRA ONESTEP RT-QPCR MASTER MIX


OSMULTIPLEX-10ML PrecisionMULTIPLEX OneStep RT-qPCR Master Mix 10ml: 10 x 1.0ml

OSMULTIPLEX-R-10ML PrecisionMULTIPLEX OneStep RT-qPCR Master Mix with ROX* 10ml: 10 x 1.0ml

OSMULTIPLEX-LR-10ML PrecisionMULTIPLEX OneStep RT-qPCR Master Mix with low ROX** 10ml: 10 x 1.0ml

OSMULTIPLEX-iC-10ML PrecisionMULTIPLEX OneStep RT-qPCR Master Mix for Bio-Rad machines 10ml: 10 x 1.0ml

OSMULTIPLEX-CL-10ML PrecisionMULTIPLEX OneStep RT-qPCR Master Mix for Roche LightCycler 1.0-2.0

10ml: 10 x 1.0ml

OSMULTIPLEX-MX-10ML PrecisionMULTIPLEX OneStep RT-qPCR Master Mix for Stratagene MX machines 10ml: 10 x 1.0ml


42

PrecisionMULTIPLEX qPCR Master Mix is ideal when you have many different tests to run, or if you have a limited number of machines. This 2X master mix contains a unique formulation to maximise the processing capacity, enabling detection of up to 4 targets simultaneously.

The core components are a hot start Taq polymerase enzyme with an ammonium sulphate based buffer. Our unique formulation gives a superior quality of data with a bigger signal to noise ratio, brighter, steeper and more precise amplification plots. Stabilisers and preservatives ensure that multiple freeze-thaw cycles do not affect the performance giving you reliable, reproducible results.

Product features•At least 50% increase in throughput•Precise reproducible results when multiplexing up to 4 targets in

single well•Optimised to run on fast and standard cycling protocols•Exceptional value for money

Precision®MULTIPLEX qPCR Master Mix

Amplification graph demonstrating reliable and uncompromised detection of 4 targets using Precision®MULTIPLEX qPCR Master Mix

4-plex qPCR testing conducted on the Bio-Rad CFX instrument using Primerdesign Precision-MULTIPLEX qPCR Master Mix. A 4-way genesig Human Pathogen assay (including endogenous control primer and probe) was used to test target template mixtures prepared to model a co-in-fection sample. Triplicate tests were performed. When used in conjunction with PrecisionMULTI-PLEX qPCR Master Mix the assay demonstrated an improved amplification profile of low copy target pathogen in comparison to a standard master mix also recommended as suitable for multiplex testing.

Amplification

0

2

4

6

8

10

12

Cycles0 10 20 30 40 50

RFU

(10^

3)

PrecisionMULTIPLEX qPCR Master MixBlue (FAM) = Target pathogen 1, Green (VIC) = Target pathogen 2, Purple (Cy5) = Target pathogen 3, Red (ROX) = Endogenous control

Standard Master MixLight blue (FAM) = Target pathogen 1, Light green (VIC) = Target pathogen 2, Violet (Cy5) = Target pathogen 3, Pink (ROX) = Endogenous control

Optimised to run on fast and standard cycling

protocols

43

PRECISIONMULTIPLEX QPCR MASTER MIX


PMULTIPLEX-10ML PrecisionMULTIPLEX qPCR Master Mix 10ml: 10 x 1.0ml

PMULTIPLEX-R-10ML PrecisionMULTIPLEX qPCR Master Mix with ROX* 10ml: 10 x 1.0ml

PMULTIPLEX-LR-10ML PrecisionMULTIPLEX qPCR Master Mix with low ROX** 10ml: 10 x 1.0ml

PMULTIPLEX-iC-10ML PrecisionMULTIPLEX qPCR Master Mix for Bio-Rad machines 10ml: 10 x 1.0ml

PMULTIPLEX-CL-10ML PrecisionMULTIPLEX qPCR Master Mix for Roche LightCycler 1.0-2.0 10ml: 10 x 1.0ml

PMULTIPLEX-MX-10ML PrecisionMULTIPLEX qPCR Master Mix for Stratagene MX machines 10ml: 10 x 1.0ml


44

PrecisionMULTIPLEX OneStep RT-qPCR Master Mix can help when you have many different tests to run, or if you have a limited number of machines. This 2X master mix contains a unique formulation to maximise the processing capacity, enabling detection of up to 4 RNA or DNA targets simultaneously.

The core components are a hot start Taq polymerase and a reverse transcriptase enzyme, including an ammonium sulphate based buffer and a special additive to reduce primer-dimers. Our unique formulation gives a superior quality of data with a bigger signal to noise ratio, brighter, steeper and more precise amplification plots. Stabilisers and preservatives ensure that multiple freeze-thaw cycles do not affect the performance giving you reliable, reproducible results.

Product features•At least 50% increase in throughput•Precise reproducible results when multiplexing up to 4 targets in

single well•Optimised to run on fast and standard cycling protocols•Exceptional value for money

Precision®MULTIPLEX OneStep RT-qPCR Master Mix

Amplification graph demonstrating reliable and uncompromised detection of 3 targets using Precision®MULTIPLEX OneStep RT-qPCR Master Mix

3-plex qPCR testing conducted on the Bio-Rad CFX instrument using Primerdesign PrecisionMULTIPLEX OneStep RT-qPCR Master Mix. A 3-way genesig Human Pathogen assay (including internal extraction control primer and probe) was used to test target template mixtures prepared to model a co-infection sample. Triplicate tests were performed. When used in conjunction with PrecisionMULTIPLEX OneStep RT-qPCR Master Mix the assay demonstrated a complete rescue of low copy target pathogen in comparison to a standard one step master mix also recommended as suitable for multiplex testing.

Amplification

0

5

10

15

Cycles0 10 20 30 40 50

RFU

(10^

3)

PrecisionMULTIPLEX OneStep RT-qPCR Master MixBlue (FAM) = Target pathogen 1, Red (ROX) = Target pathogen 2, Green (VIC) = Internal extraction control

Standard OneStep Master MixLight blue (FAM) = Target pathogen 1, Pink (ROX) = Target pathogen 2, Light green (VIC) = Internal extraction control

At least 50% increase in throughput

45

PRECISIONMULTIPLEX QPCR MASTER MIX


PMULTIPLEX-10ML PrecisionMULTIPLEX qPCR Master Mix 10ml: 10 x 1.0ml

PMULTIPLEX-R-10ML PrecisionMULTIPLEX qPCR Master Mix with ROX* 10ml: 10 x 1.0ml

PMULTIPLEX-LR-10ML PrecisionMULTIPLEX qPCR Master Mix with low ROX** 10ml: 10 x 1.0ml

PMULTIPLEX-iC-10ML PrecisionMULTIPLEX qPCR Master Mix for Bio-Rad machines 10ml: 10 x 1.0ml

PMULTIPLEX-CL-10ML PrecisionMULTIPLEX qPCR Master Mix for Roche LightCycler 1.0-2.0 10ml: 10 x 1.0ml

PMULTIPLEX-MX-10ML PrecisionMULTIPLEX qPCR Master Mix for Stratagene MX machines 10ml: 10 x 1.0ml


Amplification graph demonstrating comparable amplification produced by Precision®CONTROL qPCR Master Mix

A DNA sequence based on a fragment of a viral genome was subjected to serial dilutions. These serial dilutions were spiked with Primerdesign internal extraction control template. The serial dilutions were then tested with both PrecisionCONTROL qPCR Master Mix and an alternative standard master mix. The alternative standard master mix used a genesig Advanced Kit (supplied with separate internal control primer and probe). PrecisionCONTROL qPCR Master Mix used a genesig Standard Kit (no internal control primer and probe supplied). PrecisionCONTROL qPCR Master Mix produced identical data to the comparison standard master mix, demonstrating that no loss of performance is observed with the PrecisionCONTROL qPCR Master Mix simplified experimental setup.

Amplification

0

5

10

15

Cycles0 10 20 30 40 50

RFU

(10^

3)

genesig Standard Kit with PrecisionCONTROL qPCR Master MixBlue (FAM) = Target pathogen, Green (VIC) = Internal extraction control

genesig Advanced Kit with Standard Master MixLight blue (FAM) = Target pathogen 1, Light green (VIC) = Internal extraction control

46

PrecisionCONTROL qPCR Master Mix enables a streamlined workflow for high quality qPCR results by incorporating process controls that are a critical feature of qPCR experiments. In its premixed form this 2X master mix detects the Primerdesign internal control template without the need of a separate assay.

The core components are a hot start Taq polymerase enzyme with stabilisers and preservatives to ensure that multiple freeze-thaw cycles do not affect the performance giving you reliable, reproducible results. Our unique formulation gives a superior quality of data with a bigger signal to noise ratio, brighter, steeper and more precise amplification plots.

Product features•Built in process control for high quality qPCR results•Minimises the risk of experimental error•Precise reproducible results• Increases your lab throughput•Exceptional value for money•SYBR® Green dye added free of charge• Inert blue loading dye added free of charge

Precision®CONTROL qPCR Master Mix

47

PRECISIONCONTROL QPCR MASTER MIX


PCONTROL-XX-10ML*** PrecisionCONTROL qPCR Master Mix 10ml: 10 x 1.0ml

PCONTROL-R-XX-10ML*** PrecisionCONTROL qPCR Master Mix with ROX* 10ml: 10 x 1.0ml

PCONTROL-LR-XX-10ML*** PrecisionCONTROL qPCR Master Mix with low ROX** 10ml: 10 x 1.0ml

PCONTROL-iC-XX-10ML*** PrecisionCONTROL qPCR Master Mix for Bio-Rad machines 10ml: 10 x 1.0ml

PCONTROL-CL-XX-10ML*** PrecisionCONTROL qPCR Master Mix for Roche LightCycler 1.0-2.0 10ml: 10 x 1.0ml

PCONTROL-MX-XX-10ML*** PrecisionCONTROL qPCR Master Mix for Stratagene MX machines 10ml: 10 x 1.0ml

*Recommended for ABI 7000, 7300, 7700, 7900, StepOne and StepOnePlus - If your machine is not specified please enquire **Recommended for ABI 7500, ViiA7 and QuantStudio - if your machine is not specified please enquire ***For the complete catalogue number, please replace the XX with the type of internal control fluorophore required: FAM, Cy5, VIC, TAMRA, or ROX.

Temperature, Celcius

Melt Curve

70

10

15

20

25

30

35

72 74 76 78 80

RFU

(10^

3)

Melt curve plot demonstrating the performance of Precision® HRM Master Mix

High resolution melting analysis was conducted using the Bio-Rad CFX instrument using primers targeting the genomic region of the Factor V Leiden (R506Q) single nucleotide polymorphism.

Primerdesign genotyping thermocycling parameters were used to amplify DNA from mock sam-ples prepared using template representing AA homozygotes and GA heterozygotes.

Using Precision HRM Master Mix produced a clear discrimination between genotypes. This demonstrates that Precision HRM supports the production of high quality data in single nucleo-tide genotyping applications.

Blue: AA Homozygote melt curve produced with Precision HRM Master MixLight Blue: GA Heterozygote melt curve produced with Precision HRM Master Mix

48

High resolution melting (HRM) analysis is the most affordable and most elegant genotyping technology available. HRM analysis differs from real-time PCR in the need for a true saturating intercalating dye to be used. This, alongside a high quality Primerdesign HRM assay, ensures sharp, tight, reproducible data.

Our Precision HRM Master Mix is a high specification mix which contains all that is required for perfect real-time PCR amplification followed by precise high resolution melting analysis. The core component is a chemically modified hot start Taq polymerase with a Magnesium Chloride based buffer. The mix is stabilised to prevent damage from multiple freeze-thaw cycles.

The Precision HRM Master Mix is available with an inert blue dye to aid pipetting accuracy.

Product features•Especially formulated for optimal HRM analysis•Supplied with high spec HRM intercalating dye•High cost saving•As good or better than leading brands•Precise, reproducible results• Inert blue loading dye added free of charge

Precision® HRM Master Mix

Especially formulated for optimal HRM

analysis

49

PRECISION HRM MASTER MIX


Precision-HRM Precision HRM Master Mix 6ml

50

High quality, robust 2X qPCR master mix and OneStep RT-qPCR master mix supplied lyophilised.

The core components are a hot start Taq polymerase enzyme and a modified MMLV reverse transcriptase enzyme with a magnesium chloride based buffer. Stabilisers and preservatives ensure that lyophilisation does not affect the performance.

Product features•Supplied lyophilised – no cold shipping required•Precise reproducible results•One product works perfectly with all real-time PCR machines

oasig lyophilised reagents represent a milestone in qPCR technology Their formulation stabilises all of the active components and allows them to be shipped and stored at room temperature. They are stable for more than 18 months at ambient temperatures. This hugely simplifies the logistics of purchasing, shipping and using the technology. Whether you are in a sophisticated laboratory in Texas or a mobile field hospital in Timbuktu we can supply complete qPCR kit and reagent packages to your door quickly and cheaply via standard shipping methods without the need for dry ice or a cold chain of any sort.

The performance of the reagents is second to none. We are confident that you will find excellent data quality and even see an improvement in data quality versus many traditional frozen master mixes.

oasig™Lyophilised 2X qPCR and oasig™Lyophilised 2X OneStep RT-qPCR Master Mix

Amplification plot demonstrating the consistency of oasig™ Lyophilised qPCR Master Mix after repeated freeze thaws

Testing was conducted on the BioRad CFX instrument using a selection of Primerdesign rat gene assays and standard genesig cycling conditions.

Two separate tubes were used to amplify the rat genes from rat cDNA. The first tube was sub-jected 10 freeze-thaw cycles (freezing conducted at -20°C) whilst the second served as a posi-tive control (kept in -20°C storage).

No significant effect on the performance of oasig Lyophilised qPCR Master Mix was observed after 10 freeze-thaws. This demonstrates suitability of oasig Lyophilised qPCR Master Mix for multiple freeze-thaw uses and illustrates that optimal performance levels will be maintained under these conditions.

Blue: oasig Lyophilised qPCR Master MixLight Blue: oasig Lyophilised qPCR Master Mix after 10 freeze-thaw cycles

Cycles

Amplification

0

0

5

10

15

20

25

30

10 20 30 40 50

RFU

(10^

3)

Supplied lyophilised – no cold shipping

required

51

OASIG LYOPHILISED MASTER MIX


oasig-standard-150 oasig Lyophilised 2X qPCR Master Mix 150 reactions

oasig-onestep-150 oasig Lyophilised 2X OneStep RT-qPCR Master Mix 150 reactions

Amplification Plot

Cycle

Rn

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 41 42 44 46 48 50

1,000,000

2,000,000

3,000,000

4,000,000

5,000,000

6,000,000

7,000,000

0

Brightwhite Clear Plastic

Overall BrightWhite vs Clear Plastic

52

The best possible real-time PCR is performed using white, opaque qPCR plates. As fluorescence is the crucial output from a real-time PCR reaction, it is essential that as much of the fluorescent information available as possible is captured. Clear plastics spray light in all directions. BrightWhite qPCR plates channels all of the fluorescent output from your reaction straight back to the detector.

When using white plastic we recommend using a PCR inert blue dye, which makes visualising your pipetting much easier on white plastic.

Product features•Precisely moulded for specific machines•White opaque plastic gives brighter, better data•Extreme uniformity of wall thickness•Truly flat plates reduce well-to-well variability•Cost effective pricing

BrightWhite Real-Time PCR qPCR Plates

At least 5 x more fluorescent output vs. clear

plastic

53

BRIGHTWHITE REAL-TIME PCR PLATES


BW-96AB1 96-well plates for ABI machines: ABI7000, ABI7300, ABI7500, ABI7700, ABI7900

10 plates

BW-FAST 96-well plates for ABI7500 Fast, ABI7900 Fast and ABI StepOne plus machines 10 plates

BW-96GEN 96-well plates for Bio-Rad, Stratagene, Eppendorf and other machines 10 plates

BW-96480 96-well plates for the Roche LightCycler 480 and Bio-Rad CFX machines 10 plates

BW-384GEN 384-well plates for a wide variety of machines, e.g. Applied Biosystems, Bio-Rad, Eppendorf, Roche, MJ Research and Whatman Biometra

10 plates

BW-ECO 48-well plates for the Illumina Eco and PrimePro 48 40 plates

BW-ADVSEAL Optical adhesive seals for BrightWhite 96 and 384 well plates 12 seals

BW-8STRIP Optical 8 strip cap seals for BrightWhite 96 well plates 120 strips

BW-ECOSEAL Optical adhesive seals for BrightWhite 48 well plates 40 seals

54

genesig Easy tips for pipettes have been robustly assessed by Primerdesign and found to represent optimal level of performance in terms of pipetting accuracy and precision.

Product features•Sterile•Certified free of RNase/DNase, Human DNA, PCR inhibitors and

Pyrogens•Micropore filter technology•CE-IVD mark for laboratory use

genesig® Easy Tips

GENESIG EASY PIPETTE TIPS

CATALOGUE NO. PRODUCT DESCRIPTION BOX SIzE

genesigEASY-P20 Pipette tips for up to 20ul, box of 10x96 Box of 10 x 96



genesigEASY-Pipettes Set of 4 genesigEASY pipettes, 5ul, 10ul, 200ul and 500ul Set of 4 - 5ul, 10ul, 200ul and 500ul

55

Ultrapure molecular

biology grade water

RNase or DNase contamination in the water used for molecular biology assays can lead to experimental errors or inconsistencies. Our high quality RNase/DNase Free Water provides peace of mind when conducting sensitive molecular biology assays such as qPCR.

Product features• Ultrapure molecular biology grade water• RNase and DNase free• DEPC-treated, autoclaved, and filtered through a 0.2-micron filter• Suitable for applications such as qPCR

RNASE/DNASE FREE WATER


RNase/DNase free water RNase/DNase Free Water 15ml (10 x 1.5ml aliquots)

RNase/DNase Free Water suitable for molecular biology

56

The ideal positive control for your experiment. The BioBank is a high quality source of cDNA validated for use in real-time PCR experiments. The cDNA is reverse transcribed from high quality, DNase treated RNA, from a variety of tissues and cell cultures, using an optimised blend of oligo(dT) and random nonamer primers. BioBank cDNA is therefore free of genomic DNA and PCR inhibitors and covers the widest possible range of RNA and mRNA transcripts in the specified tissue or cell line. BioBank cDNA is useful for expression profiling of newly identified genes and also as a positive control for real-time PCR.

Positive control primer/probe are supplied with the kits which detect 18S ribosomal RNA. The 18S control primers are especially formulated to work under all recommended real-time PCR cycling conditions; i.e. TaqMan® cycling and SYBR® Green cycling conditions will all work perfectly with the kit.

Product features•High quality cDNA•Validated for use in real-time PCR•Free from PCR inhibitors•Guaranteed signal•Reverse transcribed using optimal oligo(dT)/random

nonamer blend

BioBank Control cDNA

Kit contents•250ng of gDNA-free cDNA (minimum 25 reactions)•Positive control 18S primer and probe set•RNase/DNase free water

57

BIOBANK CONTROL cDNA

HUMAN


cDNA-hu-ge Generic BioBank cDNA pooled from a collection of human tissues and cells 250ngcDNA-hu-ti BioBank cDNA from a specific human tissue* 250ng

cDNA-hu-cl BioBank cDNA from a specific human cell line* 250ng

cDNA-hu-pr BioBank cDNA from a specific primary cell* 250ng

MOUSE


cDNA-mo-ge Generic BioBank cDNA pooled from a collection of mouse tissues and cells 250ngcDNA-mo-ti BioBank cDNA from a specific mouse tissue* 250ng

RAT


cDNA-ra-ge Generic BioBank cDNA pooled from a collection of rat tissues and cells* 250ngcDNA-ra-ti BioBank cDNA from a specific rat tissue* 250ng

* Please contact us or visit our website for the latest list of available tissues/cell lines and primary cells.

58

Negative test results are only valid when the suitability of the reagents, template and protocols have also been demonstrated. The Primerdesign Real-Time PCR Internal Control Kit contains either control RNA or DNA. This can be spiked in to your sample prior to nucleic acid extraction or diluted and spiked directly into your real-time PCR reaction. A positive signal of around Cq 25 indicates that your experimental procedure is working well.

The Internal Control kit can be multiplexed or used in parallel with your test samples. The assay is primer limited and based on an obscure nucleic acid sequence so will not therefore interfere with the detection of your target sequence.

Product features•Perfect internal control•Hydrolysis (TaqMan® style) probe•Available with a wide range of fluorophores•Primer limited to prevent interference with target

gene primers

Kit contents•Lyophilised internal control primers/hydrolysis

(TaqMan® style) probe (150 reactions)• Internal control DNA or RNA template•RNase/DNase free water

Real-Time PCR Internal Control Kit

Perfect internal control

59

REAL-TIME PCR INTERNAL CONTROL KIT

RNA


INT-RNA-FAM RNA Internal PCR control with FAM dye 150 reactionsINT-RNA-CY5 RNA Internal PCR control with Cy5 dye 150 reactions

INT-RNA-YY RNA Internal PCR control with Yakima Yellow (VIC substitute) dye 150 reactions

INT-RNA-TAMRA RNA Internal PCR control with TAMRA dye 150 reactions

INT-RNA-PULSAR RNA Internal PCR control with Pulsar 650 dye 150 reactions

DNA


INT-DNA-FAM DNA Internal PCR control with FAM dye 150 reactionsINT-DNA-CY5 DNA Internal PCR control with Cy5 dye 150 reactionsINT-DNA-YY DNA Internal PCR control with Yakima Yellow (VIC substitute) dye 150 reactionsINT-DNA-TAMRA DNA Internal PCR control with TAMRA dye 150 reactionsINT-DNA-PULSAR DNA Internal PCR control with Pulsar 650 dye 150 reactionsINT-DNA-CY5-LL Suitable for use with ultra-fast master mixes 150 reactions

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Quantification of genomic DNA is useful for forensic research and also as a normalising signal for quantitative PCR and cell counting. The Primerdesign genomic DNA detection assays are sensitive down to 25pg of genomic DNA.

No more RT negative controls: The Primerdesign genomic DNA (gDNA) detection assays targets only gDNA and not cDNA and is the perfect alternative to the use of a ‘reverse transcription negative control’.

These primer sets are designed against single copy sequences within the genome. This sequence is not within any known gene.

Product features•Genomic DNA specific•Sensitive to pg quantities of DNA•Novel replacement for ‘RT negative control’

Detection kit contents•Optimised primer/hydrolysis (TaqMan®-style) probe mix (150

reactions) RNase/DNase free water

Quantification kit contents•Optimised primer/hydrolysis (TaqMan®-style) probe mix (150

reactions) •gDNA positive control template • Internal extraction control DNA • Internal extraction control primer/probe mix (150 reactions) •RNase/DNase free water

Genomic DNA Detection Kits

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gDNA-hu Human genomic DNA detection kit 150 reactionsgDNA-mo Mouse genomic DNA detection kit 150 reactions

gDNA-ra Rat genomic DNA detection kit 150 reactions

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gDNA–hu-q Human genomic DNA quantification kit 150 reactionsgDNA-mo-q Mouse genomic DNA quantification kit 150 reactions

gDNA-ra-q Rat genomic DNA quantification kit 150 reactions

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Sensitive to pg quantities

of DNA

Quantification of Y chromosome DNA is useful for forensic research. The Primerdesign Y chromosome DNA detection assays are sensitive down to 25pg of target DNA.

Product features•Y chromosome specific•Sensitive to pg quantities of DNA

Detection kit contents•Optimised primer/probe mix (150 reactions)•RNase/DNase free water

Quantification kit contents•Optimised primer/probe mix (150 reactions)•gDNA positive control template• Internal extraction control DNA• Internal extraction control primer/probe mix (150 reactions)•RNase/DNase free water

Y CHROMOSOME DETECTION KITS

WITH TAQMAN® STYLE PROBE


yDNA-hu-DD Human Y chromosome DNA detection kit with hydrolysis (TaqMan® style) probe 150 reactionsyDNA-hu-DD-q Human Y chromosome DNA quantification kit with hydrolysis (TaqMan® style)

probe150 reactions

Y Chromosome Detection Kits

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Primerdesign are experts in molecular diagnostic assay development. We have conducted many successful projects with leading diagnostic and biotechnology companies. Please contact us to enquire about how we can assist with the following services (or visit http://www.primerdesign.co.uk/assay-development):

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Primerdesign productsPrimerdesign’s impressive >600 genesig® diagnostic product range and proprietary reagents (master mixes and positive controls) are available on either an OEM basis or for distribution. For more information please visit www.primerdesign.co.uk.

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Experts in the design of complex molecular diagnostic kitsAssay optimisation, verification and validationLyophilisation of final product

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Technical Bulletins

So this section of our catalogue is designed to help you succeed in your real-time PCR and to avoid some of the common pitfalls. We have tried to walk you through some of the classical queries and questions that our customers run in to.

By sharing this knowledge and experience with you we hope that your understanding of real-time PCR will improve and so will your data.

More training and educationWe frequently spend time sharing our expertise in real-time PCR amongst the scientific community. If you would be interested in one of our team coming to your organisation to present a genuinely helpful, free, lunch-time seminar on real-time PCR then please do contact us: [email protected]

Alternatively check out our free online webinars at www.primerdesign.co.uk/learn/free-qpcr-webinars

We believe that we have failed you if your experiments fail. We love it when people get fantastic data and make exciting discoveries using our kits and reagents. So we place a lot of emphasis on making sure that every experiment you do with our kits gets you meaningful, scientifically accurate data.

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Technical bulletins66 01 Real-Time PCR Basic Principles

70 02 Probe vs SYBR® Green

72 03 RNA Extraction and Sample Preparation

74 04 Reverse Transcription

76 05 Designing High Quality Primers

78 06 Troubleshooting when using Intercalculating Dyes

82 07 Normalisation

84 08 Standard Curves – Measuring Primer Efficiency

88 09 The Delta Cq method – Relative Quantification

90 10 Standard Curves – Measuring Absolute Copy Number

92 11 The MIQE Guidelines

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PCR or the Polymerase Chain Reaction has become the cornerstone of modern molecular biology the world over. Real-time PCR is an advanced form of the Polymerase Chain Reaction that maximizes the potential of the technique.

To understand real-time PCR it is easier to begin with the principles of a basic PCR: PCR is a technique for amplifying DNA. There are 2 reasons why you may want to amplify DNA. Firstly you may want to simply create multiple copies of a rare piece of DNA. For example a forensic scientist may want to amplify a tiny piece of DNA from a crime scene. More commonly however you may wish to compare 2 different samples of DNA to see which is the more abundant. DNA analysis requires amplification in order for there to be enough DNA to give a detectable signal for quantification. If you amplify both samples at the same rate, you can calculate which sample had the highest copy number of the target of interest to begin with.

A thermostable polymerase enzyme drives a PCR. This polymerase will synthesize a complementary sequence of bases to any single strand of DNA providing it has a double stranded starting point.

This is very useful because you can choose which gene you wish the polymerase to amplify in a mixed DNA sample by adding small pieces of DNA complimentary to your gene of interest. These small pieces of DNA are known as primers because they prime the DNA sample ready for the polymerase to bind and begin copying the gene of interest.

During a PCR, changes in temperature are used to control the activity of the polymerase and the binding of primers.

To begin the reaction the temperature is raised to 95°C. At this temperature all double stranded DNA is “melted” in to single strands:

The temperature is then lowered to ~60°C. This allows the primers to bind to your gene of interest:

Technical bulletin 01

Real-Time PCR Basic Principles

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Thus the polymerase has somewhere to bind and can begin to copy the DNA strand:

The optimal temperature for the polymerase to operate is 72°C so at this point the temperature is sometimes raised to 72°C to allow the enzyme to work faster.

There are now twice as many copies of your gene of interest as when you started:

This temperature change is repeated through around 40 ‘cycles’. Thus one copy becomes 2, 2 become 4, 4 become 8, and so on until billions of copies are created.

After amplifying your gene it is possible to run the amplified DNA out on an agarose gel and stain it with a dye which makes it visible. The brighter the visible band, the more copies of your target you have created.

Real-Time PCRThis same principle of amplification is employed in real-time PCR. But instead of looking at bands on a gel at the end of the reaction, the process is monitored in “real-time”. Literally, the reaction is placed in to a real-time PCR machine that watches the reaction occur with a camera or detector.

There are a many different techniques that are used to allow the progress of a PCR reaction to be monitored but they all have one thing in common. They all link the amplification of DNA to the generation of fluorescence which can simply be detected with a camera during each PCR cycle. These different techniques are discussed on page 70. Hence, as the number of gene copies increases during the reaction, so the fluorescence increases.

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Real-time PCR has many benefits over the old fashioned approach:• Firstly it gives you a look in to the reaction. You can literally see

which reactions have worked well and which have failed.• The efficiency of the reaction can be precisely calculated. • There is also no need to run the PCR product out on a gel after the

reaction as the melt curve analysis effectively does this for you. • The greatest advantage of all however, is that real-time PCR

data can be used to perform truly quantitative analysis of gene expression. In comparison, old fashioned PCR was only ever semi-quantitative at best.


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IntroductionWhen designing a real-time PCR experiment a significant decision is choosing the correct detection chemistry for your application. There are two major options to choose from. Most people use either an intercalating dye (e.g. SYBR® Green) or a hydrolysis probe based detection solution (e.g. TaqMan®). Both technologies are designed to generate fluorescence during the PCR, which allows your real-time PCR machine to monitor the reaction in “real time”.

SYBR® Green (or other intercalating dye)SYBR® Green is by far the most commonly used intercalating dye. There are others available but it’s very likely that SYBR® Green is the one you have heard of. All of these dyes operate via a simple mechanism.

The dye is fluorescent in it’s own right but in the presence of double stranded DNA, the dye intercalates with (binds in to) the DNA double helix. This alters the structure of the dye and causes it to fluoresce more. So very simply as the PCR creates more DNA, more dye can bind and more fluorescence is generated.

Hydrolysis (TaqMan® style) Probes Probes are fluorescently labelled DNA oligonucleotides. They are designed to bind downstream of one of the primers during the PCR reaction and to give a fluorescent signal during the reaction. The 5’ end of the probe is labelled with a fluorescent reporter molecule. FAM is a green reporter and is the most commonly used but there are others such as VIC, JOE, CY5 etc which emit light at different wave lengths and can be read through separate detecting channels. On the 3’ end of the probe is a quencher molecule. This is another molecule that effectively quenches the output from the reporter. Therefore, when the reporter and quencher are physically close to one another the overall level of fluorescent output is low.


Probe vs SYBR® Green Choosing the correct detection chemistry for your experiment

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During the PCR the probe binds downstream of the primer. The probe is then cleaved by the polymerase enzyme during the reaction. By cleaving the probe the reporter and quencher are separated which means that the quencher no longer has its effect over the reporter and the level of fluorescence increases. This means that with every cycle of PCR more probe is cleaved and more fluorescence is generated.

Things to Consider: CostCost is an important part of any experimental design. It is more expensive to use a hydrolysis probe than an intercalating dye. This means that if you have an experiment looking at lots of different genes or targets then you will probably want to choose an intercalating dye as your detection chemistry. However, if you only have a few targets of interest and you know that they will be your focus, you should choose to use a hydrolysis probe.

Things to Consider: SpecificityHydrolysis probes: Overall, hydrolysis probes give more convenient data to work with than intercalating dyes. This is because of their inherent specificity. When you get a signal from a hydrolysis probe you can be sure that the signal has come from genuine amplification of your target sequence. It is only possible to get a signal when the primers and the probe bind in the correct place, to the correct target. So no post run analysis is required to confirm the correct target has been amplified. This means the data is inherently reliable and simple to work with.

Intercalating dyes: the weakness of intercalating dyes is that they are non specific. If your PCR amplifies the wrong target, or even

more than one target, you will still get an amplification plot that looks identical to a genuine signal. Intercalating dyes will bind to and report on any double stranded DNA that is formed during the reaction regardless of what it is. This means that additional analysis is required to be certain of your results. This analysis is in the form of a ‘melt curve’ (sometimes called ‘dissociation curve’). Melt curves are discussed in detail on page 78. They require more time and more experience to interpret correctly. It is crucial to interpret the melt curve along side the amplification plot in order to work with your data.

Things to Consider: Target AbundanceBecause of its inherent lack of specificity, intercalating dyes are less effective when your target of interest is rare. When a target is rare the primers are more likely to form primer dimers or to bind to the wrong target. Even the very best primers will eventually form a primer dimer and give a false signal after enough cycles of PCR if no authentic target of interest is present. However, when there is plenty of your target of interest present then intercalating dyes work very nicely.

So as a rule of thumb, if your target of interest is rare (Cq values >30), then using an intercalating dye may well be troublesome and a hydrolysis probe is recommended. But if your target of interest is abundant (Cq values <30) then using an intercalating due will be perfectly sufficient.

Related productsCustom designed real-time PCR primers pg 24-27PrecisionPLUS Master Mix pg 32-33

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IntroductionMany experiments centre around extracting RNA from a sample (cells, biopsy, blood etc) then performing a reverse transcription reaction to create cDNA to act as a template for the real-time PCR reaction. Successful extraction of pure, high quality RNA is essential to achieve good quality data. Put simply, poor RNA will guarantee you poor quality data.

Columns or Phenol?The most common RNA extraction protocols use either small anion exchange columns (e.g. QIAGEN columns) to facilitate RNA extraction or use a phenol based precipitation method (e.g. Trizol). Both can work well but the column based kits are far more convenient and tend to result in a higher quality and yield of RNA. The phenol based methods are cost effective but are fiddly and rely on a high degree of benchside experience and skill in order to yield good quality RNA. Our recommendation is therefore is to use a column based solution. Invest in good RNA at this stage and everything else downstream will become much more successful.

RNA QualityIt is impossible to stress enough how important good quality RNA is for successful real-time PCR. RNA is inherently fragile. Your skin is covered with RNases designed to protect you from RNA viruses. These will destroy RNA in the blink of an eye. So it is essential to follow good laboratory practices when working with RNA. Set up a designated RNA working area with a PCR hood. Wear a lab coat and gloves, and always keep RNA on ice when not frozen.

Once you have extracted RNA you need to be certain it is of a good quality before you proceed with a relatively expensive real-time PCR protocol. If your RNA is of a poor quality then you should discard it and start again as it will only yield poor quality data downstream.

To check RNA quality there are a few options available. Nanodrop machines (or similar) are commonly found in many laboratories these days and will tell you about the quantity and the purity of your RNA. However, they do not give any information about RNA integrity which is equally critical. For this, the ideal tool is a Bioanalyser (micro fluidic electrophoresis machine). Several such machines are available on the market. They work by assessing the relative abundance and integrity of 18S and 28S ribosomal RNA and will give you a simple quality score for your RNA. If you do not have access to one of these then you can simply run a small amount of your RNA on a 1% agarose gel and stain with ethidium bromide (or equivalent). When viewed under UV, good quality RNA will show two distinct


RNA Extraction and Sample Preparation

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bands (18S and 28S ribosomal RNA), where as degraded RNA will appear as a smear on the gel leading from the ribosomal bands to the smaller fragments at the top of the gel.

DNase TreatmentDuring RNA extraction it is very likely that you will incidentally co-extract gDNA alongside the RNA. This will cause problems downstream if it is not removed so a quick and simple DNase treatment should be carried out. This needs to be done thoughtfully as it is crucial not to damage the RNA during this process. We recommend the use of our DNase treatment kit (see page 10).

Related productsgenesig® Easy DNA/RNA Extraction Kit pg 04-05 genesig® Easy LysoBead Direct-to-PCR Extraction Kit pg 08-09 Precision DNase kits pg 10-11

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IntroductionReverse transcription (RT) is a simple and elegant process that converts RNA into cDNA thus creating a suitable template for real-time PCR. The RNA is primed by the addition of single stranded DNA primers that bind to the RNA. Where primed, the reverse transcriptase enzyme can bind to the RNA and synthesise a complimentary strand made of DNA.

The best reverse transcription protocols use thermostable enzymes capable of working at relatively high temperatures (approx 55C°). At these higher temperatures enzymes work faster and there is less tangling of the RNA. This results in a faster more efficient reaction.

Priming StrategyIt is important to put some thought into the priming strategy that you use in your RT reaction. Different strategies are suitable for different applications.

Random primers Random primers are short (typically 6-9 bases in length) DNA primers of a random sequence. They are designed to bind all over your RNA allowing reverse transcription of the entire sequence. A feature of random primers is that they will prime ribosomal RNA as well as messenger RNA. For most people it is not necessary to prime rRNA as it is mRNA that is the template of interest. rRNA accounts for around 90% of all the RNA in an extraction. This means that if you are not interested in rRNA targets, then 90% of the enzyme activity is wasted in

this instance, resulting in an inefficient reverse transcription of the mRNA you are interested in. In this scenario it is better to use one of the alternative priming strategies described below. Random primers are suitable however, if you are looking to measure rRNA expression, working with partially degraded RNA or working with bacteria and viruses where the mRNA do not have polyA tails.

Oligo(dT) primersOligo(dT) primers are simply a short string of T’s. These are designed to bind to the polyA tail of mRNA, thus creating a priming site at the 3’ end of the gene to which the RT enzyme can bind and begin reverse transcription. The advantage of this approach is that it is mRNA specific. This makes for a more efficient RT reaction in most cases as the enzyme activity is focused on mRNA and ignores the abundant rRNA. Typically a switch to Oligo(dT) priming can result in a 2-4 fold increase in RT efficiency and this improves your subsequent real-time PCR data.


Reverse Transcription

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Oligo(dT) primers are not suitable if your RNA is degraded, if you are hoping to measure rRNA expression, or when working with species where the mRNA does not have a polyA tail.

Gene specific primersGene specific priming is the gold standard way to perform reverse transcription. An RT primer is designed at the same time as your real-time PCR primers and binds specifically to the same region as your real-time PCR primers. When this primer is used for the RT step then the enzyme activity is focused only on your gene of interest resulting in a maximally efficient reverse transcription reaction. This approach is particularly useful if your gene of interest is rare and you are trying to achieve the maximum possible signal strength to improve your data.

The perceived downside of gene specific RT priming is that you can only measure one target of interest in the resultant cDNA. i.e. you cannot return to the cDNA at a later date and study the expression of other genes. This is true if only one gene specific primer is used. But there is no reason why up to 20 or 30 gene specific primers for all your targets of interest cannot be pooled and then used as an RT priming strategy. This still focuses the enzyme activity on only a few targets. Compared to other random priming strategies that prime thousands of transcripts, this is still a very focused approach and will result in better real-time PCR data.

Related productsPrecision RT kits pg 12-13

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High quality primers are critical to obtaining good quality data. The reverse statement is also true. Poor primers will always produce inconsistent and poor quality data.

Designing and optimising high quality primers can be a time consuming and onerous task. We are the best in the World at designing primers so the simplest and often most cost effective route to take is to ask us to design and optimise your primers for you. We can have your perfect primers back to you in as little as two weeks.

If you do decide to have a crack at designing primers yourself there are a wide range of issues that need to be given careful consideration in order to meet the high standards for quantitative PCR. This is a brief overview of the key points.

In order to succeed, primers must be:• Specific to one target• Active in amplifying only one PCR product• Highly efficient at initiating PCR

BioinformaticsIn order to do quantitative PCR for a single target, the primers must bind only to that one target. This may sounds very obvious but in practice there are often closely related transcripts or pseudo-gene transcripts present in the genome that must be excluded from detection. The best way to analyse the bioinformatics specificity of the primers is to do sequence comparison with the entire amplicon (the full length of the PCR product which includes the primers). The most useful resources are the publically available NCBI databases

(http://blast.ncbi.nlm.nih.gov/Blast.cgi)

Priming SpecificityThe priming specificity can only be tested experimentally by performing a real-time PCR reaction on your cDNA. To do this you need to include an intercalating dye ( e.g. SYBR® Green) in the reaction and perform a melt curve analysis (see page 78). A single melting peak of the correct predicted temperature indicates that only the one PCR product of interest has been amplified by the primers.

Priming Efficiency Priming efficiency can be measured by performing a standard curve (see page 84). However, ensuring good performance is a matter of good primer design. Poor priming efficiency is caused by poor primer to template binding kinetics and by priming artefacts that can occur with poorly designed primers. The biggest factor that can affect the efficient binding of primers to the target template is the thermodynamic structure of the single DNA strand to which the primers bind. Single stranded DNA strands have the intrinsic property to fold into complex hairpin structures. If these structures occur strongly at the site of the primer binding then the efficiency of binding is reduced leading to poor PCR priming efficiency. Ideally the template structure should be modelled using the latest software. However some fairly crude online tools are available for free. The Mfold program can be used to indicate if template structure is likely to be a problem (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form).


Designing High Quality Primers

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The other main factor that can reduce the priming efficiency, particularly at the limits of PCR detection, is primer dimer formation. If the primers themselves have complimentary sequences then they can bind to each other leading to the production of short PCR products. Amplification of primer dimers robs the intended template of primers, enzyme and other reaction components thus reducing the PCR priming efficiency. Primers must be checked for cross homology so that primer interactions are reduced.

Testing your PrimersHistorically in PCR people have spent a lot of time optimising reaction conditions to make bad primers work better. We don’t believe that a bad set of primers should be fixed in this way. It often leads to a more complicated and harder to interpret set of data. The best advice we can you give you is that if your primers are not working well for you with minimal optimisation then throw them away and start over. Alternatively let us use our expertise and state of the art processes to deliver a high quality validated design straight into your reaction.

Related productsCustom designed real-time PCR primers pg 24-27

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IntroductionIn order to use intercalating dyes in real-time PCR successfully it is crucial to understand that data interpretation involves a combination of analysis of the amplification plots and the corresponding melt curves.

Melt curvesMelt curves are a simple tool that essentially replace the running of post PCR gels that were required with standard old fashioned end point PCR. All real-time PCR machines are capable of running an automatic melt curve (sometimes called dissociation curve) as part of the real-time PCR protocol on the machine.

The PCR product is gradually heated from around 55°C to 95°C and fluorescent data is collected during the process. At the beginning of the programme, the DNA is double stranded, bound to SYBR® Green and therefore highly fluorescent. As the PCR product is heated up, eventually the melting temperature of the PCR product is reached and the two DNA strands separate releasing the SYBR® Green in to

solution. This results in a sudden drop in fluorescence. Subsequently all of the PCR product is melted and the level of fluorescence plateaus off again.

Most real-time PCR machines take this data and present it more conveniently. Instead of plotting absolute fluorescence, the rate of change of fluorescence is plotted. Therefore, the sudden change in fluorescence seen at the melting temperature of the PCR product is visualised as a sharp peak against a relatively flat background where only a small change of fluorescence is occurring.

So in a perfect reaction a single distinct peak indicates that a single distinct PCR product has been amplified. It is analogous to seeing a distinct single band on a gel. The melting temperature of the PCR product can be predicted from the length of the amplicon and the proportion of G and C bases. So it is possible to check that the “correct” product has been amplified based upon the predicted melting temperature of the PCR product. Most real-time PCR amplicons will have a melting temperature of around 80-95°C when using our PrecisionPLUS Master Mix


Troubleshooting when using Intercalating Dyes (SYBR® Green)

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If you see an imperfect melt curve you will need to interpret the result in order to understand the problem.

As a rule of thumb a melt peak with a Tm less than 80°C is most likely to be caused by primer dimers. Another clue is that primer dimers tend to melt over a wider temperature range resulting in a less sharp peak with a wider base. A primer dimer is formed where the forward and reverse primers bind to one another creating a polymerase binding site. After enough cycles of PCR the polymerase will eventually succeed in extending this product and amplifying it thus giving a false signal. This problem typically occurs when the target DNA is very rare, or even absent. In this scenario, with no authentic template to bind to, primers are much more likely to form dimers.

All of our primers are designed to have minimum propensity to form primer dimers, and in the presence of authentic template all of them will give specific amplification of the target of interest. However, even with the very best primers it is still sometimes possible to achieve a primer dimer signal after 33+ cycles of PCR when the

target of interest is absent. This is not necessarily a problem as long as you are capable of interpreting the melt curve correctly so that you ignore data from primer dimers and only work with data from authentic amplification plots.

If you are getting primer dimer signals that result in amplification plots with a Cq value of 30 or less then the primers are unsuitable and should be redesigned.

More often than not a melt curve with a melting temperature higher than the target of interest is caused by amplification on gDNA. An amplicon may be designed to span an intron to prevent detection of gDNA. However, if the intron is small it is sometimes possible to amplify across the intron creating a large PCR product. This is why it is best practice to DNase treat your RNA prior to the reverse transcription step as this problem is eliminated then and there.

Alternatively, a peak with an unexpected melting point may indicate that your primers are mis-priming and amplifying the wrong product. Mis-priming on a more G/C rich template will give you an extra peak

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at a higher temperature just as mis-priming on a more A/T rich template will give you an extra peak at a lower melting temperature.

A signal in your no template controlAll good real-time PCR experiments have a no template control (NTC). This is where the DNA is replaced with water. A signal in the NTC indicates something is not perfect and should be investigated. That said, it is not always a disaster.

NTC signals tend to come from one of two places:

Primer dimers As described above primer dimers are easy to spot on a melt curve. They are only a problem if they are interfering with the real signals from your real targets of interest. If a set of primers gives a good signal with a single melt peak when the target of interest is present but a primer dimer signal when it is absent this data is still interpretable. The limits of detection are defined by the point at which a primer dimer signal appears. As long as this is understood the data can still be scientifically valid.

If however, the primer dimer signal is present alongside your gene of interest signal - i.e. two peaks on your melt curve, then the data is uninterpretable and the primers will need to be redesigned.

PCR product contamination Real-time PCR generates billions of copies of your target of interest with every reaction. This PCR product is a massive potential contaminant for your future experiments. For this reason you should try never to open a PCR reaction after the PCR if you can help it. Aerosols from opening a reaction plate can and will contaminate your laboratory. If you do need to open a PCR plate make sure it

is in a different laboratory well away from where you set up your real-time PCR reactions. The danger with PCR product is that it is the perfect template for your real-time PCR primers. Therefore, if it ends up contaminating a future experiment it can lead to false positive results. This is why a NTC is so important. Amplification in the NTC of a product with a melting temperature the same as your target of interest shows that you have PCR product contamination in your laboratory/reagents. This is a very common problem but is not necessarily a disaster. Most laboratories will have a degree of persistent contamination with the targets of interest that are most frequently amplified in the lab. Again, the secret is to interpret your data correctly when contamination is present. If the contamination is so bad that the NTC Cq values are similar your “real” signal in your “real samples” then the data is not interpretable as you can’t be sure if the signal in your samples is biologically derived or as a result of contamination. However, if your real signals are greater than 6 cycles earlier than your NTC signals then you can be confident that your data is reliable and is not affected by this small amount of PCR product contamination.

If significant contamination has been identified you need to get rid of it quickly before repeating work. You can spend a great deal of time performing exclusion experiments to identify the precise cause, but PCR contamination is fiendishly hard to track down as the number of potential entry points is very high. It is usually much quicker to discard all currently reagents and primers for that target and decontaminate the lab. Pay particular attention to door handles, buttons, pipettes and surfaces. Start afresh by running a NTC control with no positive samples included so that you can be absolutely sure your reagents are now clean. This approach may initially seem wasteful but it can save a lot of human recourses and machine time.


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Limit of DetectionsIntercalating dyes (e.g. SYBR® Green) work well when your target of interest is abundant. However, they run into problems when the target of interest is rare. Primer dimers and non specific binding events become much more common when the target gene is rare which makes using intercalating dyes problematic. Thus as a general rule of thumb the limits of detection when using an intercalating dye sit at around cycle number 30. If your target of interest takes greater than 30 cycles to cross the cycle threshold line then we would recommend switching to a probe based detection chemistry such as a hydrolysis (TaqMan®) probe.

Beyond this rule of thumb you can define your own limits of detection for your particular experiment by identifying the number of cycles of PCR where your signal becomes unreliable i.e. where primer dimers are present in your melt curves and/or when your NTC signals are less than 6 cycles later than ‘real’ signals.

Related productsCustom designed real-time PCR primers pg 24-27 qbase+ pg 20-21 PrecisionPLUS Master Mix with SYBR Green pg 32-33

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Why Normalise?During the preparation of cDNA for real-time PCR analysis there is significant potential for small errors to accumulate. For example, differences in sample size, RNA extraction efficiency, pipetting accuracy and reverse transcription efficiency will all add variability to your samples. Thus, if you are going to perform relative gene expression between samples, it becomes essential to answer the question; “how much cDNA is really present in these samples?”

Strategies to ConsiderWhen comparing one cDNA sample to another researchers have employed a number of strategies to try and “normalise” their data;

• Standardising sample size. Its good practice to try and use similar sized samples (e.g. a similar number of cultured cells or similar sized biopsy), however this is insufficient as a normalisation strategy because it does not account for the cumulative errors that can occur in cDNA preparation

• Normalising to genomic DNA. This strategy can be effective but is impractical because most RNA preparation protocols deliberately eliminate the presence of DNA

• Normalising to total RNA. RNA quantification using a Bioanalyzer (Agilent) is a useful step. The analysis provide useful information about the quality of your RNA but again does not account for the cumulative errors that can occur in cDNA preparation

• Normalising to a reference (housekeeping) gene. Normalising to a stably expressed reference gene that is representative of the cDNA concentration in a sample is the most

commonly used normalisation approach. The reference gene is subject to the same errors in cDNA preparation as the gene of interest so makes an excellent normalising control. However, careful and strategic selection of the most stably expressed reference gene is essential. Random selection of a reference gene can add large unpredictable error to your analysis

Reference (housekeeping) Gene SelectionNormalising real-time PCR data to the expression of a reference (housekeeping) gene is an excellent and practical strategy. However, reference genes are not stably expressed in all scenarios. Using the wrong reference gene for normalisation will make your data scientifically invalid. It is essential therefore to establish which reference genes are the most stably expressed in your particular experimental model. Your model is unlikely to be identical to anyone else’s so it is important to do this step for yourself.

Quick, Simple Reference Gene Selection with geNorm™

geNorm is a piece of software designed to establish the most stably expressed reference genes for any particular model. Following measurement of a number of candidate reference genes in 10 or so samples by real-time PCR the user inputs the data into geNorm. The software then carries out analysis of the relative ratio of expression of each reference gene. Thus the candidate reference genes are ranked in order of stability.

The software also provides useful information about the optimal number of reference genes that need to be averaged in order to


Normalisation

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achieve the very best normalisation strategy. The geNorm software is a proven application and the original article is one of the most viewed articles on BioMed Central of all time, with over 2000 citations in other peer reviewed articles.

The geNorm™ KitIn collaboration with Biogazelle (Belgium), Primerdesign can provide geNorm kits for anyone working with human, rat or mouse samples plus a huge range of other species. The kit comprises 6 or 12 high quality reference gene real-time PCR assays and access to the latest geNorm software. The geNorm kit provides the simplest, quickest and most affordable route to establishing the best normalisation strategy for your real-time PCR research.

FAQ’sQ: Which samples should I use in my geNorm analysis?

A: The most important thing to ensure is that all of the experimental

conditions are represented fairly. e.g. treated and untreated samples. You need equal numbers of each. geNorm treats all samples the same and does not know which are treated and which are untreated so it is up to you as the user to represent them both equally so that geNorm can identify the genes most stably expressed across the experiment.

Q: How many samples should I use in my geNorm analysis?

A: A minimum of 8, but the more you run the better and more meaningful the data. 20 would be ideal.

Related productsgeNorm kits pg 16-19 Reference gene assays pg 22-23

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What is a Standard Curve?A standard curve is a tool used in real-time PCR to measure the efficiency with which a given set of primers amplifies the target gene; the amplicon. In an ideal experiment a set of primers will work with 100% efficiency. This means that with every cycle of PCR the amount of amplicon present is multiplied by 2. If a primer set is working with less than 100% efficiency then the amount of amplicon is multiplied by less than 2 with each cycle of PCR.

To perform a standard curve reaction the user takes their template DNA and performs a serial dilution. Typical this will be a 10 fold serial dilution over 6 points.

Point 1: neat DNAPoint 2: 1:10 dilutionPoint 3: 1:100 dilutionPoint 4: 1:1000 dilutionPoint 5: 1:10000 dilutionPoint 6: 1:100000 dilution

The user then uses 5µl of this as the template for their real-time PCR. This results in 6 Cq values (sometimes known as Ct values).

Then a graph of dilution vs Cq value is plotted:

The slope of this graph can then be converted into a percentage efficiency value. Most real-time PCR machine software packages do this automatically. But for simplicity’s sake it is easy to remember that a slope of -3.3 indicates an efficiency of 100%. If the slope is less than this then the priming efficiency is less than 100% (e.g. Slope of -4.2 = efficiency of 73%)


Standard Curves – Measuring Primer Efficiency

85

A good set of primers will report an efficiency between 90-110%, so you want to see a slope of between -3.1 and -3.6 in an ideal experiment.

The Problem with the Standard Curve Method The standard curve is the best method that we have available to measure priming efficiency. However, it can be problematic. The reason for this is that the efficiency that a standard curve reports is influenced by more than just the priming efficiency. This leads to much frustration as different primer sets can report wildly different efficiencies on different days on different templates. There are two other major factors that alter the result obtained from a standard curve. It is important to understand both:

1. The template itself can massively alter the apparent result from a standard curve experiment. If the template in use is less than perfect, cDNA from degraded RNA for example, then this can result in a poor quality standard curve. The result will indicate the primers are performing with poor efficiency when in fact the primers may

be perfectly good. Similarly, if the template contains impurities (proteins, lipids etc) this can result in a very different level of efficiency being reported. These impurities act as PCR inhibitors.

2. The abundance of the target gene can also hugely alter the result achieved from a standard curve experiment. When the target of interest is rare it is impossible to get a sufficient number of good data points as the dilution leads to the assay reaching the limits of detection. Thus if the target gene is abundant it tends to appear that a primer set is performing with high efficiency where as the same set of primers will report very low priming efficiency when using template with a low level of target gene present.

Because the results obtained from a standard curve are so variable dependent on these additional factors, it makes it a somewhat scientifically flawed way to discover the true priming efficiency of a set of primers. If you have performed the standard curve method yourself using gDNA or cDNA you will no doubt have noticed how difficult it is to obtain consistent results from your standard curves.

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A solution to the ProblemThe solution to this problem is to standardise the template that is used. Biologically derived gDNA or cDNA are by their very nature, very variable. Instead it is best practice to use a copy of the amplicon cloned in to a vector. This provides a clean and stable template from which to create a standard curve. Such a tool will give reliably reproducible data day after day and most importantly will help you to very simply understand the real efficiency with which your primers are working. We can provide a copy number standard at 2 x 105 copies per microlitre if you require.

FAQsQ. My standard curve indicates that my primers are priming

with an efficiency of greater than 100%. How is this possible?

A. A priming efficiency of greater than 100% is of course beyond the theoretic limits of amplification since a PCR reaction can only double the amount of product in each PCR cycles and no more. However, a reported priming efficiency of greater than 100% can be obtained from a standard curve. This results from a flattening of the standard curve gradient. In other words the amplification plots have been compressed together. When using SYBR® Green as the detecting chemistry, compression of the standard curve at late PCR cycles is commonly due to primer dimer formation, so make sure you check the melt curves before making a final judgement. It is always a good idea to remove outlying points as

these can massively alter the reported efficiency.

Q. What are the acceptable limits of priming efficiency to work within?

A. Ideally you want a reported efficiency of 90-110%. This indicates that your primers are priming with an efficiency close to 100%. This makes them suitable for the delta Cq method of data analysis. Life is simple in this instance.

Q. What do I do if my priming efficiency is outside of the range 90-110%?

A. First of all be certain that your primers really are outside of this range. Consider all the things discussed in this bulletin. Then if your primers really do fall outside of this range then you have 2 options. You can try and correct for this poor efficiency within your calculations - the Pfaffl method - (this method should be used with caution as without a thorough expert understanding it can actually multiply errors rather than reducing them). Or you can throw out the primers and start again. We believe that the second option is the preferable one. It is more accurate to work with good quality primers than to try and correct for poor primers using mathematics.

Related productsqbase+ pg 20-21 Custom designed positive control, for more information please contact [email protected]


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IntroductionIn the vast majority of experiments the correct and most useful way to analyse gene expression data is by relative quantification. i.e. to asses how gene expression levels change between one subject group relative to another. This is different to absolute quantification but far simpler to execute and often far more accurate in its conclusions. The best and simplest method to calculate relative quantification is using the so called delta Cq method (also called the delta Ct method in the literature).

A Simple Principle When a PCR reaction is working well, with 100% efficiency, then every cycle of PCR doubles the amount of target DNA in the tube. Based on this principle it is correct to conclude that if a PCR reaction reaches ‘take off point’ one cycle earlier than another PCR reaction then the first reaction contained twice as much target DNA as the second at the beginning of the reaction. It is this simple principle that underlies how real-time PCR can be used to produce accurate quantitative data.

One cycle earlier = twice the amount of target DNA present in the tube at the beginning of the reaction.

Expressing this MathematicallyTo express this mathematically is simple. The difference between two samples is known as the delta Cq value (Delta is the Greek letter D and stands for ‘Difference’). So to compare two samples you simply subtract the Cq value of one sample away from the other to find the difference between the two.


The Delta Cq Method – Relative Quantification

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Then you use the ‘delta Cq’ formula to convert this number to a relative ‘fold change’ in gene expression.

Relative level of expression = 2(delta Cq)

So in this example 21 = 2 fold greater expression in sample A than sample B.

In this example the delta Cq is 11 (29-18=11)

211 = 2,048 fold greater expression in sample A than sample B

The Pfaffl MethodA very well cited variation of the delta Cq method is the so called Pfaffl method. Named after the esteemed author of the publication that described the approach (A new mathematical model for relative quantification in real-time RT–PCR. Michael W. Pfaffl, Nucleic Acids Res. 2001 May 1; 29(9): e45.)

The delta Cq formula 2delta Cq is based around the fact that every cycle

of PCR results in 2 fold increase in PCR product. If a PCR is less than 100% efficient then this is not true and the delta Cq formula is not appropriate to use. Pfaffl suggests in this instance that the formula be adjusted according to the real efficiency of the reaction in question.

e.g. If the PCR is only 80% efficient each cycle of PCR results in a 1.8 fold increase in PCR product so the correct formula to use is 1.8delta Cq

This technique is appropriate to use if your are forced into using an inherently inefficient set of primers. It relies on a very accurate analysis of priming efficiency to make it reliable, as any inaccuracy in calculating priming efficiency will result in errors being multiplied in downstream analysis. Because of this huge potential for error we recommend that if your primers are less than 90% efficient it is better practice to discard the primers and design new ones rather than to try and fix poor primers with mathematical corrections.

Any primers with a priming efficiency between 90% and 110% are suitable for use with the traditional delta Cq method, and we will guarantee a priming efficiency of greater than 90% for any primers that we design for you, thus making them very simple to work with.

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Why Measure Absolute Copy Number?Standard curves can be used in a real-time PCR experiment to quantify precisely the number of copies of a target DNA molecule present in a given sample*. In a lot of experiments this is an unnecessary thing to do. It is important to ask yourself if there is any scientific benefit to know the precise number of copies of a target present. Does it have any biological meaning in your experimental model to know that, for example, there are 1000 copies per microlitre of your target present. More often than not it is more relevant to measure the fold change in expression of a target in a treated group vs a control group, or a healthy sample vs. diseased sample. In this case it’s more appropriate to use a technique such as the delta Cq method. However there are circumstances where knowing the precise number of copies of target present in a sample is crucial. For example when using real-time PCR as a diagnostic tool to measure HIV viral load in a patient’s blood stream, as knowing the precise level of target present has a real clinical relevance as it effects the dose of anti retroviral drug to be administered.

How to Perform a Standard Curve to Measure Absolute Copy NumberTo perform a standard curve reaction to measure absolute copy number you first need a sample that contains your target at a known concentration. The best way to achieve this is to use a Primerdesign positive control which is a synthetic version of your target amplicon cloned in to a vector. It is then carefully calibrated and supplied at a precise concentration of 2 x 105 copies per microlitre. Thus if you use 5 microlitres in a real-time PCR reaction it will contain 1 million copies.

The user takes their template DNA and performs a serial dilution. Typically this will be a 10 fold serial dilution over 6 points.

Point 1: 2 x 105 copies per microlitrePoint 2: 2 x 104 copies per microlitrePoint 3: 2 x 103 copies per microlitrePoint 4: 200 copies per microlitrePoint 5: 20 copies per microlitrePoint 6: 2 copies per microlitre

The user then uses 5µl of this as the template for their real-time PCR. This results in 6 Cq values (sometimes known as Ct values).

Then a graph of copy number vs Cq value is plotted:


Standard Curves – Measuring Absolute Copy Number

91

Then the user can compare the Cq value that is achieved in an unknown sample with the standard curve and very simply “read off” the number of target molecules present in the sample.

Then the data can be expressed as a concentration relative to the original sample.

E.g. Copies per microlitre of serumE.g. Copies per cellE.g. Copies per gram of tissue

*n.b. Standard curves can also be used to measure the efficiency with which a set of primers is working (see page 84). The focus of this technical bulletin however is on the use of standard curves to measure absolute copy number.

Related productsqbase+ pg 20-21 Custom designed positive control, for more information please contact [email protected]

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IntroductionDuring the past decade, several high-profile cases of faulty research have been linked to inconsistent real-time PCR techniques and experiments. In April 2009, Professor Stephen Bustin and an international team of nine scientists, joined forces and developed a set of guidelines for the publishing qPCR results. The resulting ‘MIQE guidelines’ outline the minimum information required to publish quantitative real-time PCR data with scientific integrity.

If you want to do real-time PCR properly, to produce scientifically relevant results and to get your data published then this is the one paper you should read:

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Clin Chem. 2009 Apr;55(4):611-22.

Primerdesign is cited in the MIQE guidelines because we share the same philosophy on primer sequences as the authors. We have always provided the primer sequences with our custom designed assays as we believe that this is crucial to perform research with integrity.

We have included a summary of the MIQE guidelines herein to act as

a convenient guide for you. Don’t be daunted by the list. 90% of the items are common sense and you are probably already doing them. However, there may be a few things in there that will make a real improvement to the quality of your data.

We strive to make all of our products compliant with the MIQE guidelines and will always be on hand to guide and advise you in producing real-time PCR data of the highest quality.

MIQE Checklist Keya All essential information (E) must be submitted with the

manuscript. Desirable information (D) should be submitted if available. If primers are from RTPrimer DB, information on qPCR target, oligonucleotides, protocols, and validation is available from that source.

b FFPE, formalin-fixed, paraffin-embedded; RIN, RNA integrity number; RQI, RNA quality indicator; GSP, gene-specific priming; dNTP, deoxynucleoside triphosphate.

c Assessing the absence of DNA with ano–reverse transcription assay is essential when first extracting RNA. Once the sample has been validated as DNA free, inclusion of ano–reverse transcription control is desirable but no longer essential.

d Disclosure of the probe sequence is highly desirable and strongly encouraged; however, because not all vendors of commercial predesigned assays provide this information, it cannot be an essential requirement. Use of such assays is discouraged.


The MIQE Guidelines

93

MINIMUM INFORMATION FOR PUBLICATION OF QUANTITATIVE REAL-TIME PCR EXPERIMENTS

MIQE CHECKLIST FOR AUTHORS, REVIEWERS, AND EDITORS.a

ITEM TO CHECK IMPORTANCE

EXPERIMENTAL DESIGN

Definition of experimental and control groups E

Number within each group E

Assay carried out by the core or investigator’s laboratory? D

Acknowledgment of authors’ contributions D

SAMPLE

Description E

Volume/mass of sample processed D

Micro dissection or macrodissection E

Processing procedure E

If frozen, how and how quickly? E

If fixed, with what and how quickly? E

Sample storage conditions and duration (especially for FFPEb samples) E

NUCLEIC ACID EXTRACTION

Procedure and/or instrumentation E

Name of kit and details of any modifications E

Source of additional reagents used D

Details of DNase or RNase treatment E

Contamination assessment (DNA or RNA) E

Nucleic acid quantification E

Instrument and method E

Purity (A260

/A280

) D

Yield D

RNA integrity:method/instrument E

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RIN/RQI or Cq of 3’and 5’ transcripts E

Electrophoresis traces D

Inhibition testing (Cq dilutions, spike, or other) E

REVERSE TRANSCRIPTION

Complete reaction conditions E

Amount of RNA and reaction volume E

Priming oligonucleotide (if using GSP) and concentration E

Reverse transcriptase and concentration E

Temperature and time E

Manufacturer of reagents and catalogue numbers D

Cqs with and without reverse transcription Dc

Storage conditions of cDNA D

qPCR TARGET INFORMATION

Gene symbol E

Sequence accession number E

Location of amplicon D

Amplicon length E

Insilico specificity screen (BLAST, and soon) E

Pseudogenes, retropseudogenes or other homologs? D

Sequence alignment D

Secondary structure analysis of amplicon D

Location of each primer by exon or intron (if applicable) E

What splice variants are targeted? E

qPCR OLIGONUCLEOTIDES

Primer sequences E

RTPrimer D B identification number D

Probe sequences Dd


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Location and identity of any modifications E

Manufacturer of oligonucleotides D

Purification method D

qPCR PROTOCOL

Complete reaction conditions E

Reaction volume and amount of cDNA/DNA E

Primer, (probe), Mg2+, and dNTP concentrations E

Polymerase identity and concentration E

Buffer/kit identity and manufacturer E

Exact chemical composition of the buffer D

Additives (SYBR Green I, DMSO and so forth) E

Manufacturer of plates/tubes and catalog number D

Complete thermocycling parameters E

Reaction set up (manual/robotic) D

Manufacturer of qPCR instrument E

qPCR VALIDATION

Evidence of optimisation (from gradients) D

Specificity (gel, sequence, melt or digest) E

For SYBR Green I, Cq of the NTC E

Calibration curves with slope and y intercept E

PCR efficiency calculated from slope E

CIs for PCR efficiency or SE D

r2 of calibration curve E

Linear dynamic range E

Cq variation at LOD E

CIs throughout range D

Evidence for LOD E

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If multiplex, efficiency and LOD of each assay E

DATA ANALYSIS

qPCR analysis program (source, version) E

Method of Cq determination E

Outlier identification and disposition E

Results for NTCs E

Justification of number and choice of reference genes E

Description of normalisation method E

Number and concordance of biological replicates D

Number and stage (reverse transcription or qPCR) of technical replicates E

Repeatability (intra assay variation) E

Reproducibility (interassay variation, CV) D

Power analysis D

Statistical methods for results significance E

Software (source, version) E

Cq or raw data submission with RDML D


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Human (Homo sapiens):• Actin, beta (ACTB)• Glyceraldehydes phosphate dehydrogenase (GAPDH)• Ubiquitin C (UBC) • Beta-2-microglobulin (B2M) • Phospholipase A2 (YWHAz) • Ribosomal protein L13a (RPL13A) • 18S Ribosomal RNA (18S) • Cytochrome c-1 (CYC1) • Eukaryotic translation initiation factor 4A (EIF4A2) • Homo sapiens succinate dehydrogenase (SDHA) • DNA topoisomerase I (TOP1)• ATP synthase, (ATP5B)• Excision repair cross-complementing rodent repair deficiency (ERCC6) • Ubiquitin-conjugating enzyme E2D 2 (UBE2D2) • Ubiquitination factor E4A (UBE4A)• Ecto-NOX disulfide-thiol exchanger 2 (ENOX2)• PR domain containing 4 (PRDM4)• Selenocysteine lyase (SCLY)• tRNA-yW synthesizing protein 1 homolog (S. cerevisiae) (TYW1)• Ring finger protein 20 (RNF20)• Chromosome 14 open reading frame 133 (C14orf133)

Mouse (Mus musculus):• Actin, beta, cytoplasmic (ACTB)• Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)• Ubiquitin C (UBC)• Beta-2 microglobulin (B2M)• Phospholipase A2 (YWHAz)• Ribosomal protein L13a (RPL13A)• Calnexin (CANX)• Cytochrome c-1 (CYC1)• Succinate dehydrogenase complex, subunit A (SDHA)• 18S Ribosomal RNA (18S)

• Eukaryotic translation initiation factor 4A2 (EIF4A2)• ATP synthase subunit (ATP5B)• Adaptor-related protein complex 3 (Ap3d1)• Casein kinase 2, alpha prime polypeptide (Csnk2a2)• Cell division cycle 40 homolog (Cdc40)• F-box and WD-40 domain protein 2 (Fbxw2)• F-box protein 38 (Fbxo38)• HIV TAT specific factor 1 (Htatsf1)• MON2 homolog (Mon2)• PAK1 interacting protein 1 (Pak1ip1)• zinc finger protein 91 (zfp91)

Rat (Rattus Norvegicus):• 18S Ribosomal RNA (18S)• ATP synthase, (ATP5B)• Topoisomerase I (TOP1)• Malate dehydrogenase 1 (MDH1)• Cytochrome c-1 (CYC1)• Calnexin (CANX)• Ribosomal protein L13 (RPL13)• Tyrosine 3-monooxygenase (YWHAz)• Beta-2 microglobulin (B2M)• Ubiquitin C (UBC)• Glyceraldehyde-3-phosphate dehydrogenase (GAPD)• Actin, beta (ACTB)• Nucleoporin like 2 (Nupl2)• Similar to XPA binding protein 1 (LOC688393)• Staufen RNA binding protein homolog 1 (Stau1)• F-box and WD repeat domain containing 4 (Fbxw4), mRNA• CCCH-type with G patch domain (zgpat)• Integrin-linked kinase-associated serine/threonine phosphatase 2C (Ilkap)• FtsJ methyltransferase domain containing 2 (Ftsjd2)• Tomm22 nuclear gene encoding mitochondrial

protein (Tomm22)

AppendixOur catalogue of reference (housekeeping) genes includes (but not limited to):

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Primerdesign™ is a trademark of Primerdesign Ltd.

The PCR process is covered by US Patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG.

SYBR® Green is a registered trade mark of Molecular Probes Inc. ABI, ABI PRISM® GeneAmp® and iCycler™ is a registered trademark of Bio-Rad Laboratories, Rotor-Gene is a trademark of QIAGEN. LightCycler™ is a registered trademark of the Idaho Technology Inc. GeneAmp®, TaqMan® and AmpliTaqGold® are registered trademarks of Roche Molecular Systems, Inc.

The purchase of the Primerdesign™ reagents cannot be construed as an authorisation or implicit licence to practice PCR under any patents held by Hoffmann-LaRoche Inc.

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Primerdesign is part of the Novacyt Group of companies

FM559592 MD606750

We are always delighted to help with any aspect of your real-time PCR research. Please feel free to contact us for free advice or technical support.

Primerdesign Ltd York House School Lane Chandlers Ford United Kingdom SO53 4DG

Telephone: +44 (0)23 8074 8830 Fax: +44 (0)870 836 2155Orders: [email protected]: [email protected] support: [email protected]

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