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Sample to Insight
Practical Hints and New Solutions for Successful Real-Time PCR Studies
Dr. Ina Scheuerpflug, Director LS Foundation, Strategic Marketing
Cover Page 2
1Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Maximizing Real-Time PCR Results: Sample to Insight
2
Two-Part Webinar Series
Part 1: Practical Hints and New Solutions for Successful Real-
Time PCR Studies
Part 2: Critical Factors for Successful Real-Time PCR, Multiplex
PCR
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Maximizing Real-Time PCR Results: Sample to Insight
3
Two-Part Webinar Series
Part 1: Practical Hints and New Solutions for Successful Real-
Time PCR Studies
Part 2: Improving Accuracy and Reproducibility in your Real-Time
PCR Results: Novel In-Process Monitoring Tools
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Legal disclaimer
4
QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis, prevention, or
treatment of a disease.
For up-to-date licensing information and product-specific disclaimers, see the
respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and
user manuals are available at www.QIAGEN.com or can be requested from
QIAGEN Technical Services or your local distributor.
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
5
RNA: Critical steps for real-time PCR analysis
Improved methods for cDNA synthesis
Real-time PCR essentials
New real-time PCR solutions
1
2
3
4
Cells /Tissues
RNAStabilization
RNAPurification
ReverseTranscription
Real-TimeDetection
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
6
RNA: Critical steps for real-time PCR analysis
1.1 Stabilization of RNA patterns and RNA quality
1.2 RT enzyme efficiency
1.3 Priming strategy
1.4 Genomic DNA contamination
Improved methods for cDNA synthesis
Real-time PCR essentials
New real-time PCR solutions
1
4
Cells /Tissues
RNAStabilization
RNAPurification
ReverseTranscription
Real-TimeDetection
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
What happens to RNA ex vivo?
7Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Principles of RNA stabilization
8
Title only (a)
Requirements
• Stop induction of new RNA transcription (e.g., stress response)
• Prevent regulated turnover of mRNA
• Prevent nonspecific degradation of RNA, either enzymatically (due to release of nucleases
from specific cell types or compartments), or chemically (pH, temperature, etc. dependent)
Approaches
Agents that bind directly to nucleic acids
Agents that lyse cells
Agents that inhibit, denature and/or precipitate nucleases (proteins in general)
Fixation
Preserve morphology (usually with no consideration of effects on RNA)
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Requirements for stabilization of blood and tissue samples
9
Blood and tissue samples have different stabilization technology
requirements
Blood contains only solitary cells but has a high protein content
Diffusion rates of stabilizing agent no problem
Agents that precipitate or cross-link proteins inappropriate
Blood volume is not limited in principle (container is limited)
One-step stabilization technology required
Tissue contains connected cells
Diffusion rates of the stabilizing agent are critical
Thickness of the tissue sample is limited
Multi-step technology applicable as tissue piece transferrable
Protein precipitation or cross-link is not a problem in principle
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Collection and stabilization solutions for different sample types
10Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Critical factors for RNA purification
11
Quality of the purified RNA: integrity and purity Quality of starting sample material
Inactivation of RNases
Co-purification of potential inhibitors e.g., for the RT-step
Protein (nuclease) contamination
gDNAcontamination
RNA yield Efficient cell/tissue lysis
mRNA content of total RNA is only 1–5%
Purification of total RNA and small RNA (e.g.,miRNA)
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Different lysis steps for different material
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Lysis: Protocol dependent on sample type
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
• Mechanical disruption of cells/tissue (e.g., TissueLyser)
• Lysis of cells and nuclei by chaotropic salts
• Inactivation of RNases by chaotropic salts in lysis buffer
• Proteinase for lysis of tough samples (i.e. muscle)
◦ requires conditions where proteinase is active but RNases are not
• QIAzol (phenol-guanidine based):
◦ lysis of tough samples, and removal of contaminants by organic extraction very
versatile, but more work, hazardous reagent
Sample to Insight
Silica absorption protocol
13
Lysis: Protocol dependent on the sample type
Bind nucleic acids to silica: High concentrations of chaotropic salts, low pH,
+ alcohol
Wash the silica membrane: Removal of proteins, inhibitors and chaotropic
salts
Elute nucleic acids from silica membrane: Water or low-salt buffer pH 7–8
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Choosing the right RNA purification product / protocol
14
Is the RNA purification procedure appropriate for the amount of starting material?
Overloading usually results in lower purity, lower yields
With small samples it is important to use an efficient RNA purification method
RNA purification from large samples:
Provides a pool of RNA that can be used for multiple experiments
May be required to get sufficient amounts of RNA from samples that have low RNA
content
High binding capacity – usually also implies higher dead volume
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Guidance: RNA extraction kit targeted to your starting material
15
If you would like to order the wheel for your lab just send us an email
Download the selection wheel at: www.qiagen.com
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
New RNeasy Kit development: RNeasy Plus Universal Kits
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Purification of total RNA from any tissue type using gDNA Eliminator solution
• One-for-all tissue solution
• Time-saving integration of RNeasy and QIAzol technologies
• Fast non-enzymatic removal of genomic DNA
• Pure RNA for use in all downstream applications
• Optimized protocols enable purification of high-quality RNA from any type of tissue,
even difficult-to-lyse tissues.
• QIAzol Lysis Reagent included for lysing fatty tissue and other types of tissue, and RNeasy
spin columns for purifying high-quality RNA.
Kits are available in two formats:
RNeasy Plus Universal Mini Kit enables purification of RNA from up to 50 mg tissue
RNeasy Plus Universal Midi Kit enables purification of RNA from up to 250 mg tissue.
The RNeasy Plus Universal Mini Kit can be automated on the QIAcube.
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Effect of phenol on UV absorbance
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Phenol contamination imitates higher RNA content of the sample
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
18
RNA: Critical steps for real-time PCR analysis
1.1 Stabilization of RNA patterns and RNA quality
1.2 RT enzyme efficiency
1.3 Priming strategy
1.4 Genomic DNA contamination
Improved methods for cDNA synthesis
Real-time PCR essentials
New real-time PCR solutions
1
4
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Critical factors for an efficient RT step
19Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
• Quality of the RNA starting material
◦ Phenol contamination
◦ Alcohol contamination
◦ Salt contamination
◦ Protein inhibitors
• Affinity to the RNA to avoid secondary structure effects
• Choice of priming method
◦ Random priming vs. Oligo dT priming
• Genomic DNA contamination
• Sequences near the 5' end effect the cDNA yield
• Use of internal controls is critical
RT enzyme efficiency is influenced by:
Sample to Insight
High efficiency and sensitivity – Why?
20
QuantiNova Reverse Transcriptase
Highly efficient transcription for sensitive detection even of low abundance targets
Novel Reverse Transcriptase - use of a wide range of RNA amounts (10 pg – 5 µg)
High affinity for RNA - leads to high sensitivity
Up-scaling option for larger input volumes (up to double volume)
Successful use even for difficult templates
Plus RNase inhibitor
gDNA removal buffer
Efficient removal of gDNA (>1000 fold reduction) is integrated in the protocol
RT-Primer Mix
Optimized mix of oligo-dT and random primers
Internal control
Optional use of internal control included to monitor cDNA synthesis efficiency
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
21
RNA: Critical steps for real-time PCR analysis
Improved methods for cDNA synthesis
Real-time PCR essentials
New real-time PCR solutions
3
Cells /Tissues
RNAStabilization
RNAPurification
ReverseTranscription
Real-TimeDetection
1
4
2
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
QuantiNova Reverse Transcription Kit – Protocol
22
• With unique Internal Control RNA
• With gDNAremoval
Fast and convenient protocol:
1. RNA (potentially contaminated with gDNA)
Add gDNA Removal Buffer (incl. RNase Inhibitor)
Optionally spike in Internal Control RNA
2 min 45°C
2. Synthesize cDNA
Add 5 µl RT-Master Mix
3 min 25°C, 10 min 45°C, 5 min 85°C
cDNA (incl. IC cDNA, if applicable)
Stable storable, colorable with QN Yellow Template Dye
3. Stop reaction (95°C, 3 min)
Get cDNA without gDNAcontamination!!!
cDNA synthesis procedure in only 20 min
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
QuantiNova Reverse Transcription Kit – Internal Control
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QuantiNova IC reliably indicates inhibition or failure of RT or qPCR reaction
Reliable in-process monitoring of RT and qPCR performance
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
QuantiNova Reverse Transcription Kit – gDNARemoval
24
Efficient, optional gDNA removal prevents CT shifts caused by DNA
contamination
Precise mRNA quantification even if exon spanning primers cannot be used
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Effect of primer choice
25
10 kb transcript (amplicon 2kb and 6kb away from 3´end)
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Summary: QuantiNova Reverse Transcription Kit
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High sensitivity due to novel enzyme
Synthesize cDNA and remove gDNA in 20min
Integrated gDNA removal step
Reverse transcription from all regions due to primer mix
(5’region)
Internal control
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
27
RNA: Critical steps for real-time PCR analysis
Improved methods for cDNA synthesis
Real-time PCR essentials
• Different quantification strategies
• Probe technology
• Factors influencing PCR specificity
New real-time PCR solutions
Cells /Tissues
RNAStabilization
RNAPurification
ReverseTranscription
Real-TimeDetection
4
3
1
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
PCR kinetics
28
Little or no increase in PCR product yield
Half-life of PCR enzyme
Inhibition of polymerase by end products
Starvation of PCR substrate
Generation of PCR product is
directly proportional to input
target molecules/PCR cycle
Start of PCR
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
CT = thresholdcycle
29
Cycle number at which the fluorescent signal reaches the detection threshold
Time point when the fluorescence signal can be detected by the real-time cycler
Start of PCR
DNA conc./fluorescent signal
has reached the fluorescence
detection threshold of the real-
time cycler
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Quantification methods in real time PCR
30
Comparison of differential gene expression in different samples:
Quantification
Relative Quantification Absolute Quantification
Target mRNA copies relative to an
internal RNA, e.g., GAPDH or rRNATarget mRNA copies relative to an external
standard and per cell or unit mass
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Absolute quantification
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Determines absolute copy number of sample
CT value of unknown sample compared to standard curve of known standard
Important: Amplification efficiencies of standard and target are equivalent
Log amount of standard
Initial concentration of unknown sample
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Relative quantification using ∆∆CT
32
Direct comparison of CT values
Important: both PCR reactions have same PCR efficiency
Compares ∆CT of unknown sample with ∆CT of calibratorsample
∆∆CT = ∆CT (calibrator) - ∆CT (sample)
2 – ∆∆CT = x-fold expression level of GOI in comparison to calibrator
Livak, K.J. and Schmittgen, T.D. (2001) Methods 25, 402.
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Relative quantification using ∆∆CT
33
∆CT1 (control) - ∆CT2 (sample)=
∆∆CT
2 –∆∆C
T = Expression level of TNFin
comparison to HPRT
HPRT = Hypoxanthin Phosphoribosyltransferase
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Endogenous reference gene
34
Choose a suitable gene for use as a reference or endogenous control.
An endogenous control gene‘s expression level should not differ between samples.
Comparison of CT values of a target gene and endogenous control gene allows the
target gene expression level to be normalized to the amount of input RNA or cDNA
Use of an endogenous control gene corrects for variation in RNA content and reverse-
transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA
sample, variation in nucleic acid recovery and differences in sample handling
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Endogenous reference gene
35
More information at:
http://normalisation.gene-quantification.info/
https://www.qiagen.com/de/spotlight-pages/newsletters-and-magazines/articles/endogenous-controls/
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
36
RNA: Critical steps for real-time PCR analysis
Improved methods for cDNA synthesis
Real-time PCR essentials
• Different quantification strategies
• Factors influencing PCR specificity
• Probe technology
New real-time PCR solutions
Cells /Tissues
RNAStabilization
RNAPurification
ReverseTranscription
Real-TimeDetection
4
3
1
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Different amplicon detection methods
37
Sequence-nonspecific detection with fluorescing dyes
SYBR Green
Sequence-specific probes
HybProbe
TaqMan Probes
Molecular Beacons, MGB Probes, Scorpions...
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
SYBR Green chemistry
38
Detection of all double-stranded DNA molecules
Detection of specific and nonspecific PCR products
dsDNA + free dye
(weak fluorophore)
Binds minor groove
(fluorescence ,000x)
dsDNA binding dye shows increased fluorescence when binding to the minor
groove of the DNA
This dye is excited at 497 nm and emits at 520 nm
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Annealing – the most critical step for high PCR specificity
39
Specific annealing
Specific product
High yield
High sensitivity
Due to high primer concentration
Nonspecific product (e.g., primer-dimer)
Low yield
Low sensitivity
Inaccurate quantitation
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
The melting curve shows unspecific product
40
Fluorescence drops when DNA melts
Melting point is determined as the maximum in the negative 1st derivative
negative 1st derivative
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Factors influencing PCR specificity
41
Amount of starting template
Primer design
Cations contained in reaction buffer
Initial generation of artifacts by Taq DNApolymerase
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Primer design
42
Sequence:
Length of PCR product smaller than 150 bp (ideal 100–150 bp)
Avoid complementary sequences
Avoid mismatches at the 3´–end
Avoid a 3´–end T has a greater tolerance of mismatch
Avoid G/C runs at 3’ end results in stable mismatched primers
Length:
18–30 nucleotides
GC content:
40–60% – High GC content increases the stability of primer/template
Tm = 2°C x (A+T) + 4°C x (C+G)
Concentration:
0.3 µM (e.g. ABI systems, iCycler), 0.5 µM Lightcycler
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Effect of target length
43
Amplification efficiency and sensitivity drops significantly the longer the amplicon
Improved PCR efficiency and sensitivity with shorter products (Optimal length: 100–150 bp)
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Effects of different cations
44Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Effects of different cations
45
Mg2+
Stabilize the annealing process
Low Mg2+ concentration leads to specific annealing
But: The more Mg2+ ions the more nonspecific product is seen
Using NH4+ and K+
Leads to PCR results which are not influenced by Mg2+
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
NH4+ buffers destabilize non-specifically bound primers
46
Comparison of two types of PCR buffer with respect to the Mg2+ concentration:
PCR: 150 ng human genomic DNA, 0.75 kb of single-copy gene for prion protein
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Effect of PCR specificity
47
NH4+ Buffer Buffer without NH4
+
Gene:
cDNA:
Cycler:
Human BAX
100 pg – 10 ng
Rotor-Gene 3000
Primer–dimer in the NTC showing nonspecific reaction
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
48
RNA: Critical steps for real-time PCR analysis
Improved methods for cDNA synthesis
Real-time PCR essentials
• Different quantification strategies
• Factors influencing PCR specificity
• Probe technology
New real-time PCR solutions
Cells /Tissues
RNAStabilization
RNAPurification
ReverseTranscription
Real-TimeDetection
4
3
1
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Target-specific detection
49
Hydrolysis probes:
Hydrolyzed by the Taq polymerase results in separation of the reporter and the
quencher fluorochrome
TaqMan probes
Hybridization probes:
Probe is labelled with a donor fluorochrome and an acceptor fluorochrome, but not
hydrolyzed during PCR
FRET probes, Molecular Beacons, Scorpions
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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TaqMan chemistry
50Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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TaqMan chemistry
51Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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TaqMan chemistry
52Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Which dyes can be combined?
53
Check the technical specifications of your real-time PCR cycler!
Excitation spectra:
Stephen A. Bustin, A–Z of Quantitative PCR
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Agenda
54
RNA: Critical steps for real-time PCR analysis
Improved methods for cDNA synthesis
Real-time PCR essentials
New real-time PCR solutions
3
4
Cells /Tissues
RNAStabilization
RNAPurification
ReverseTranscription
Real-TimeDetection
1
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
New real-time PCR solutions
55
QuantiNova: High efficiency and sensitivity – Why?
Novel antibody-mediated hot-start mechanism
Novel additive QuantiNova Guard stabilizes a binding complex between
DNA Polymerase and the Antibody. The DNA Polymerase remains inactive in this
configuration.
Precise reaction setup with a built-in visual indicator to minimize pipetting errors.
Accurate results with robust detection of rare targets down to a single copy
Magnified throughput and speed with ultrafast cycling
Seamless performance when combined with QuantiTect Primer Assays
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Novel additive QuantiNova Guard
56
Unmatched specificity due to a novel antibody-mediated hot-start mechanism
QuantiNova Guard stabilizes the DNA Polymerase – Antibody binding complex. DNA
Polymerase remains inactive in this configuration. Within 2 minutes at 95°C, the
antibody and QuantiNova Guard are denatured and the QuantiNova DNA
Polymerase is activated, enabling PCR amplification: https://vimeo.com/87778004
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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The new built-in control minimizes pipetting errors
57
Accurate reaction setup indicated by the new built-in pipetting control:
QuantiNova SYBR Green PCR Master Mix contains an inert blue dye,
when combined with QuantiNova Yellow Template Dilution Buffer the
resulting solution turns green – indicating correct reaction setup.
Does not interfere with the reaction
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
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Summary: QuantiNova PCR Kit Chemistry
58
QuantiNova SYBR Green and Probe Kits streamline qPCR by delivering:
High specificity due to a novel antibody-mediated hot-start mechanism
Precise reaction setup with built-in visual indicator to minimize pipetting errors
No need for optimization (e.g., no search for the optimal annealing temperature
or a Mg2+ titration)
Accurate results with robust detection of rare targets down to a single copy
Magnified throughput and speed with ultrafast cycling
Seamless performance when combined with QuantiTect PrimerAssays
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
For additional information
59
You can find all product information at:
https://www.qiagen.com/de/products/life-science-
research/cancer-research/gene-expression-analysis/
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017
Sample to Insight
Thank you for attending
60
Contact QIAGEN Technical Service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
www.support.qiagen.com
Ina Scheuerpflug, Ph.D.
Dirk Schacht, Ph.D.
Questions?
Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017