Mi rna array 2013

Sample & Assay Technologies

miScript miRNA PCR Arrays

Simultaneous Detection of Genome-Wide or Pathway-Focused miRNA

Samuel Rulli, [email protected]@sabiosciences.com (US)[email protected] (international)

Sample & Assay Technologies- 2 -

Topics to be Covered

Introduction to miRNA

miScript miRNA PCR Array Overview� miRNA isolation� miScript miRNA PCR Arrays and Assays� Pre-amplification of miRNA� Data Analysis

miScript miRNA PCR Array Applications� Cancer� Development & Differentiation� Genome-Wide Discovery


What Is MicroRNA?

.Endogenously expressed small functional RNAs (19-25 nt)

.Regulate mRNA expression post-transcriptionally� mRNA degradation, but also translation inhibition

.Only partial sequence complementarity required� One miRNA can potentially regulate hundreds of mRNAs� One mRNA can be regulated by multiple miRNAs

Another layer of complexity for regulating gene expression


Canonical pathway of microRNA biogenesis

� Transcribed by RNA Polymerase II as a long primary transcript (pri-miRNAs), which may contain more than one miRNA.

� In the nucleus, Pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse III-like enzyme Drosha

� Pre-miRNAs are then exported to the Cytosol by Exportin 5

� In the cytosol RNAse III-like Dicer processes these precursors to mature miRNAs

� These miRNAs are incorporated in RISC

� miRNAs with high homology to the target mRNA lead to mRNA cleavage

� miRNAs with imperfect base pairing to the target mRNA lead to translational repression and/or mRNA degradation

Krol, J et.al., (2010) Nature Rev Genetics, 11, 597; Winter, J. et.al., (2009) Nature Cell Biology, 11, 228


miRNA genomic structure

� Intergenic miRNA genes : either monocistronic or polycistronic with a common promoter

� Intronic miRNA genes : present in the introns of protein coding or noncoding genes, can also be in clusters, transcribed by the host gene promoter

� Exonic miRNAs genes : rare and often overlap an exon and an intron of a noncoding gene

� miRNAs can be transcribed from the negative strand within or near a protein coding gene


Multiple loci can generate the same mature miRNABut are under different regulatory control

www.mirbase.org

Stem Loop CHR Overlapping transcripts CHR: Coordinat es (GRCh37)

1302-1 12 intergenic 12: 113132839-113132981 [-]

1302-3 2 intergenic 2: 114340536-114340673 [-]

1302-7 8 intergenic 8: 142867603-142867674 [-]

1302-10 15 intergenic 15: 102500662-102500799 [-]

1302-11 19 intergenic 19: 71973-72110 [+]

1302-2 1 intronic Non protein coding 1: 30366-30503 [+] sense

1302-4 2 intronic Non protein coding 2: 208133999-208134148 [-]

1302-9 9 Non protein coding 9: 30144-30281 [+]; Sense

1302-5 20 intronic Protein coding/FAM65C; intron4 20: 49231173-49231322 [-]; Sense

1302-6 7 intronic Protein coding/HDAC9; intron 1 7: 18166843-18166932 [-] ; Antisense

1302-8 9 intronic Protein coding/ch9orf174 9: 100125836-100125963 [-];

Antisense

Mature-miR-1302: UUGGGACAUACUUAUGCUAAA


miRNAs are continually being discovered

.miRNA database: www.mirbase.org

.New miRNAs continually discovered

.(release 19.0, August 2012)

� Human: 1066 (V16) 2032 (v19)� Mouse: 940 (v16) 1281 (v19)� Rat: 653 (v16) 721 (v19)

.Nomenclature� Pri-miRNA (>100nt)� Pre-miRNA (~70nt)� Mature miRNA (~20nt)


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Year

miRNA Publications

miRNA Research is an exponentially growing field

� Virtually every publication includes quantification experiments.

� Changes in miRNA correlated with gene expression changes in development, differentiation, signal transduction, infection, aging, and disease.

HighWire + PubMed Publications miRBase Entries

** Projected

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miRBase Versionmature miRNA sequences


Why Analyze miRNA Expression Patterns?

.Potential for genome-wide coverage, even though >1000 miRNAs identified

.miRNA expression profiles correlate with:� Protein & Gene Expression Levels, Biological Phenotypes

.miRNA regulated genes are involved in a variety of biological processes and diseases:

� Cancer� Development� Immunology� Aging� Heart Diseases� Neurological Diseases


Unique miRNA signatures are found in human cancer(first studies around 2005)

� miRNAs located in genomic regions amplified in cancers (e.g. miR-12-92 cluster) can function as oncogenes, whereas miRNAs located in portions of chromosomes deleted in cance rs (e.g. miR-15a-miR-16-1 cluster) can function as tumor suppressors.

� Abnormal expression of miRNAs has been found in both solid and hematopoietic tumors.

� miRNA expression fingerprints correlate with clinical and biological characteristics of tumors , including tissue type, differentiation, aggression and response to therapy.

� The race is on to develop miRNA biomarkers and ther apeutics!


Circa 2011A growing picture of miRNA dysregulation in cancer

� Deletions of miRNA-regulated genes have been detected in:� >65% of chronic lymphocytic leukemia cases, � 50% of mantle-cell lymphomas, � 16% to 40% of multiple myelomas, � 60% of prostate cancers.

� Other miRNA abnormalities have been reported in a wide variety of � Human neoplasms, including other hematologic malignancies such as

promyelocytic leukemia � Benign tumors such as leiomyoma and pituitary adenoma� Multiple types of carcinomas, including pancreatic, esophageal,

thyroid, lung, and breast� Neuroblastomas and glioblastomas


Scientific question� Well-defined and testable

Experimental sample set� Statistically meaningful number of replicates (biological replicates)

� A minimum of 3 replicates recommended� Replicates enhance the statistical power

� Inclusion of proper normalization controls & spike in controls in the analysis

Experimental testing platform� Simple, straightforward workflow� High sensitivity & specificity� A variety of PCR arrays to meet your experimental needs� Easy and simple data analysis tools� Customizable solutions & single assays� Availability of companion research tools

miRNA expression profilingKey considerations


How to Monitor miRNA Expression Levels

.Northern Blot� Very low throughput� Very time consuming and complex

.Microarrays � Profile More Sequences� Poor Sensitivity & Dynamic Range, Complicated Protocol

.Next-gen sequencing� Quantifiable?� Discover novel sequences, but need validation� Cost for routine analysis of 12+ samples (complex procedures, $$ for instrument)

.Real-Time RT-PCR � Profiles Fewer Sequences (Up to 384)� Simpler Protocol � Better Sensitivity, Specificity & Reproducibility


Topics to be Covered

Introduction to miRNA

miScript miRNA PCR Array Overview� miRNA isolation� miScript miRNA PCR Arrays and Assays� Pre-amplification of miRNA� Data Analysis

miScript miRNA PCR Array Applications� Cancer� Development & Differentiation� Genome-Wide Discovery


miScript miRNA PCR Array Systemcomplete system from isolation to published data

.miRNeasy miRNA Isolation Kit

.miScript miRNA 1st Strand cDNA Synthesis Kit� Preferentially reverse transcribe mature miRNA � Built-in external RNA control� One-step reaction

.miScript miRNA PCR Arrays and qPCR Primers� Human, mouse, rat and others: Search miRNA Primers:

http://www.qiagen.com/Products/miScriptPrimerAssays.aspx

� miScript miRNA PCR Arrays: Pathways and Genomes

.Optimized miScript SYBR® Green Master Mix

.miScript miRNA Data Analysis Excel Template & Web Portal











miRNeasy Kits: Mini and MicroFlexible protocols: total RNA or enrichment of miRN A

.High quality, pure RNA � Suitable for sensitive downstream

applications

.Rapid procedure � Streamlined protocol for low-

throughput or 96-well formats� Automated on QIAcube

Effective purification of total RNA� From a broad range of cells & tissues

.Efficient enrichment of microRNAs� miRNA enriched fraction & total RNA

separately� Co-purification of miRNA & total RNA


miRNeasy Kits: Mini and MicroFlexible protocols: total RNA or enrichment of miRN A

.miScript miRNA PCR Assays and Arrays profile mature miRNA from TOTAL RNA

� No need to separate miRNA� Use co-purified Total RNA� Best practice for mRNA and

miRNA detection� Able to use mRNA now and

archive miRNA as cDNA (increased stability and bio-banking)


miRNeasy FFPE Kit Enables purification of high quality total RNA from archival samples

500 million FFPE tissues are archived!� Tissue banks, pathology labs, biomedical research labs

Current Isolation Methods: Compromised quality and yield� Heavily fragmented� Cross-linking by formaldehyde interferes with RT� Current procedures may not purify all usable RNA, may increase

fragmentation, are often ineffective in breaking up cross-links

.miRNeasy FFPE Kit: High quality, pure total RNA� Novel method prevents cross-linked RNA from blocking

downstream applications� Optimized lysis buffer with proteinase K� RNeasy MinElute for small elution volume


miRNeasy Serum/Plasma Kit Purification of high quality total RNA from serum or plasma samples











miRNA RT-PCR Technical Difficulties

.Short sequence (21-23nt)

.Background from contaminating small RNAs (tRNA, rRNA, snRNA)

.Highly homologous� Many miRNAs have a single nucleotide difference


miScript PCR System Reverse Transcription using HiSpec Buffer


Performance: Universal RT Advantages

.Ease of Universal Reverse Transcription Reaction� Simpler setup, without primer interaction issues� Less sample necessary for analyses� Equal RT reaction for each miRNA, to ensure reproducible

expression analysis

.Comprehensive miRNA coverage� cDNA can be saved for new analyses when additional miRNA

sequences are discovered� cDNA can be pre-amplified allowing analysis from few cells or

amplification of limiting samples for miRNome analysis

� Same specificity as stem loop primers with additional flexibility











miScript miRNA PCR ArraysPathway-Focused: 84 miRNAs + 12 Controls

Cel-miR-39

miScript PCR Controls for Normalization

miRTC PPCSNORD61; SNORD68; SNORD72 SNORD95; SNORD96A; RNU6-2

RTControl

PCRControl

Spike in Control

84 miRNAs

� Cel-miR-39� Alternative data normalization using exogenously spiked Syn-cel-miR-39 miScript miRNA Mimic

� miScript PCR Controls� Data normalization using the ∆∆CT method of relative quantification

� miRNA reverse-transcription control (miRTC)� Assessment of reverse transcription performance

� Positive PCR control (PPC)� Assessment of PCR performance


miScript miRNA PCR ArraysAvailable in 96-well, 384-well, and Rotor-Disc 100 Formats

96-well 384-well

384-well (4 x 96) Rotor-Disc 100


miScript PCR ControlsStable expression and excellent amplification effic iencies

SNORD61

SNORD68

SNORD72

SNORD95

SNORD96A

RNU6-2

� Highly conserved across multiple species� Human, Mouse, Rat, Dog, Rhesus macaque

� Relatively stable expression in many tissues� Amplification efficiencies of these assays are 100%� Consistent performance in both HiSpec and HiFlex Buffers� Ideal normalizers for ∆∆CT method of relative quantification

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Brain Kidney Liver Lung Skeletal

Muscle

Testis Thymus

Mean C

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Tissue Expression

Amplification EfficienciesPrimer Hsa Mmu Rno Cfa Mml

SNORD61 103 99 95 99 101

SNORD68 101 100 93 97 99

SNORD72 94 100 93 91 97

SNORD95 102 103 98 100 106

SNORD96A 102 95 95 92 104

RNU6-2 98 98 95 99 103


miScript miRNA PCR ArraysRedefining miRNA expression profiling

� 100% validated assays� Each assay is bench validated� Each array is quality controlled

� Leading miRNome coverage

� miRNome coverage is continually updated� Validated designs on miRBase V16/V18� Assay nomenclature compatible with miRBase V19� New content added monthly for pathway focused content

� Fully customizable

Benefit of miScript miRNA PCR Arrays and Assays


miScript miRNA PCR ArraysReliable and routine miRNA expression profiling

� Human� Mouse� Rat � Dog� Rhesus macaque

Complete miRNome Arrays

Species Assays(miRBase V16)

Human 1066

Mouse 940

Rat 653

Dog 277

Rhesus macaque 469 (V18)

� Human, Mouse, Rat:� miFinder (Dog and Rhesus macaque)� Cancer PathwayFinder� Brain Cancer � Breast Cancer� Ovarian Cancer� Cell Differentiation & Development� Immunopathology � Inflammatory Response &

Autoimmunity� Neurological Development & Disease � T-Cell & B-Cell Activation � Prostate Cancer� Serum & Plasma

Pathway-Focused Arrays


miScript miRNA PCR ArraysRedefining miRNA expression profiling

� Human, Mouse, Rat:� miFinder (Dog and Rhesus macaque)� Cancer PathwayFinder� Brain Cancer � Breast Cancer� Ovarian Cancer� Cell Differentiation & Development� Immunopathology � Inflammatory Response &

Autoimmunity� Neurological Development & Disease� T-Cell & B-Cell Activation – New!� Prostate Cancer – New!� Serum & Plasma

Pathway-Focused Arrays

� miFinder 384HC� Profiles the expression of the 372 most

abundantly expressed and best characterized miRNAs in miRBase

� Serum & Plasma 384HC� Profiles the expression of 372 miRNAs

detectable in serum and plasma using the miScript PCR System

� Cancer Finder 384HC� Profiles 372 miRNAs associated with

various cancers and cancer stages

New! High Content Pathway-Focused Arrays











miScript miRNA PCR Arrays

.cDNA Synthesis (miScript II RT KIT)� 1 hours

.Load Plates (Preferably with 8-Channel Pipettors)

� 2 minutes

.Run 40 cycle qPCR Program � 2 hours

.Upload and Analyze Data� 15 minutes


miScript miRNA PCR ArraysCompatible with a wide range of instruments

� 96-Well: 7000, 7300, 7500, 7700, 7900HT, ViiA 7� FAST 96-Well: 7500, 7900HT, Step One Plus, ViiA 7� FAST 384-Well: 7900HT, ViiA 7

� Mastercycler ep realplex 2/2S/4/4S

� Mx3000p, Mx3005p, Mx4000p

� iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384� Opticon, Opticon 2, Chromo 4

� LightCycler 480

� TP-800

RotorGene Q


Performance: Reproducibility

The miscript miRNA PCR Assays are highly reproducib le, ensuring run-to-run, plate-to-plate and sample-to-s ample

reliability.


Replicates: Technical & Biological

Technical� Reproducibility of the PCR Arrays is very high� Results demonstrate that what you are seeing is a result of biology, not technique.� RTC & PPC show technical reproducibility on each plate, and comparable across plates.

Biological�Needed to verify the results are a result of biology� Need multiple samples

� At least 3 replicates per sample for statistical analysis� p values�95% Confidence Intervals


Limiting SamplesBody fluidsFFPE samplesLCM samplesFlow-sorted cellsCirculating tumor cellsFine needle biopsies

Pre-amplification for miRNA detection in limiting samples


y = 0.9973x + 0.2058

R2 = 0.9913

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Mean CT: Operator 1Mean C

T: Operator 2

� Universal reference RNA� Two operators

� Experienced operator� First time operator

� miScript real-time PCR� miFinder miScript miRNA PCR Array

miScript PreAMP PCR Kit – decrease input up to 1000XStraightforward, reproducible and robust amplificat ion


miScript PreAMP: miRNA expression profiling from li miting FFPE samplesExcellent results with a fraction of the input

y = 0.9934x - 0.1457

R2 = 0.9606

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∆CT: No PreAMP

∆CT: PreAMP

� Workflow� No PreAMP: 250 ng total RNA, HiSpec Buffer � Human miRNome miScript miRNA PCR Array (Plate 1)� PreAMP: 10 ng total RNA, HiSpec Buffer � one-tenth of cDNA, miScript PreAMP � preamplified cDNA

used for Human miRNome miScript miRNA PCR Array (Plate 1)

� Excellent correlation, PreAMP vs. No PreAMP� Preamplified cDNA provides enough material for more than 2 full human miScript

miRNome profiles. Input was only 1 ng of cDNA!


1.E-01

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2-∆CT

: Normal Serum

2-∆CT: Colorectal Cancer Serum

miRNA expression profiling using limited serum samp lesUsing the Serum & Plasma 384HC

� Workflow� 5 µl serum � 14 µl total RNA � 5 µl total RNA, HiSpec Buffer � one-tenth of cDNA, miScript PreAMP �

preamplified cDNA (0.07 µl SE) used for Human Serum & Plasma 384HC miScript miRNA PCR Array

� Conclusion: Starting with only 5 µl of serum, significant differences were detected when colorectal cancer serum was compared to normal serum

y = 0.9728x + 0.7671

R2 = 0.9757

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CT: Individual RNA Isolation from Serum

CT Mean: 3 RNA Isolations from Serum

Reproducibility 2 -∆CT: Colorectal Cancer vs. Normal











Data analysismiScript miRNA PCR Array Data Analysis Tools


.Web-Based Software� No installation needed� Tailored for each array

.Raw CT Values to Fold-Change Results� Using ∆∆CT Method

� Other methods available

.Multiple Analysis Formats� Scatter Plot� Volcano Plot� Multi-Group Plot� Clustergram

.

Downloadable Excel Templates also available

Data analysis using free softwaremiScript miRNA PCR Array Data Analysis Tools


Application Data: Cancer Biomarkers

Human Cancer miscript miRNA PCR Array:Many Cancer-Specific miRNA Are Up-regulated in a

Colon Tumor.


Application Data: Cell Differentiation & Developmen t

Human Cell Differentiation & Development miscript m iRNA PCR Array: Identifies 26 Brain- and 20 Muscle-Specific m iRNA Potentially

Regulating Tissue-Specific Gene Expression.

26 Brain-Specific miRNA

20 Muscle-Specific miRNA


Application Data: Genome-Wide Screening

miscript Human Genome miRNA PCR Array: Identifies Kn own and Novel miRNA Targets of the p53 Signaling Pathwa y

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Ct A

deno

-p53

34a 940

203 551a


Application Data: FFPE

Expression rank order of 88 cancer related miRNAs in RNA extracted from one 20µm section of a 2-year old normal human colon FFPE block using the miscript FFPE RNA Extraction Kit.


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FFPE Isolation 1

FFPE Isolation 2

FFPE Isolation 3

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Human miFinder miRNA Assay

CT Value

FFPE Isolation 1

FFPE Isolation 2

FFPE Isolation 3

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Sample

∆CT (miRTC - PPC)

∆CT < 7 indicates no inhibition of reverse-transcription

� One 5 µM FFPE section used per FFPE isolation� Each isolation is from a different section

� On average, each isolation provided enough total RNA for:

– Two full human miRNome profiles– Ten pathway-focused PCR arrays

� RT and qPCR� RT: 125 ng total RNA, HiSpec Buffer

� qPCR: Human miFinder miScript miRNA PCR Array

– 0.5 ng cDNA per well, Human miFinder PCR Array

� ∆CT (miRTC – PPC) is used to assess reverse transcription efficiency

Normal Lung

Lung Tumor

miRNA expression profiling using FFPE samples


1.E-04

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2-∆CT

: Normal Lung FFPE Tissue

2-∆CT: Tumor Lung FFPE Tissue

miRNA expression profiling using FFPE samples (cont .)

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Log2 Fold-Regulation (Tumor vs. Norm

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2-∆CT: Tumor vs. Normal Fold-Regulation: Tumor vs. Normal

� Significant differences exist between the mature miRNA expression levels of the two tissue types

� ± 2-fold [red lines] used as a cutoff for significance


1.E-05

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2-∆CT

: Normal Serum2-∆CT: Breast Cancer Serum

miR-34a

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miRNA

CT Value

Serum Isolation 1

Serum Isolation 2

Serum Isolation 3

Normal Serum 2 -∆CT: Breast Cancer vs. Normal

miRNA expression profiling using serum samples

� Workflow� 200 µl serum � 50 µl total RNA � 5 µl total RNA, HiSpec Buffer � one-half of cDNA used for 96-well

miFinder miScript miRNA PCR Array

� High reproducibility can be achieved using the miRNeasy Supplementary Protocol � Each isolation was from a different normal serum donor

� Significant differences exist between the mature miRNA expression levels of the two tissue types

� ± 2-fold [red lines] used as a cutoff for significance


miScript miRNA PCR ArraysQIAGEN PCR Array Service Core

� Total RNA Isolation: miRNeasy Kits

� Reverse Transcription: miScript II RT Kit

� qPCR: miScript miRNA PCR Arrays

� Data analysis included!

Send your samples and receive results!


SUMMARY

.miRNA Function• Regulates gene expression post-transcriptionally

.miRNA Performance• RT-PCR vs. Microarrays • Universal vs. Sequence-Specific RT• Specificity, Sensitivity & Reproducibility

.miRNA Applications• Screen cancer- or development-focused miRNA panels• Profile miRNAs by genome and follow-up with custom miRNA PCR

array panels.


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SciGlobe Advantage

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Mi rna array 2013

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