Mi rna part iii_2013

Sample & Assay Technologies

miRNA Profiling :

On the Road to Biomarker Development

The products described in this webinar are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of disease.

Jonathan Shaffer, Ph.D.

[email protected], miRNA Technologies

Research & Development

Sample & Assay Technologies Three part webinar series

Webinar 3: Profiling miRNA expression in cells, FFPE, and serum 2

miRNA and its role in human disease

Webinar 2 : Advanced microRNA expression analysis: From experimental design through data analysis

Speaker: Jonathan Shaffer, Ph.D.

Webinar 3 : Profiling miRNA expression in Cells, FFP E, and serum:On the road to biomarker development


Webinar 1 : Meeting the challenges of miRNA research :An introduction to microRNA biogenesis, function, a nd analysis


Sample & Assay Technologies Profiling miRNA expression in cells, FFPE, and seru m


Agenda

� Introduction to miRNA and disease

� Where and how do you access the information? � Samples and sample prep options

� Profiling miRNA expression using the miScript PCR S ystem� System components, options, and workflows

� Summary of the miScript PCR System

� Questions

Sample & Assay Technologies microRNA (miRNA) timeline


Natural phenomenon, discovery tool, potential bioma rker

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Discovery ofLin-4, Ambros

PTGS in plantsBaulcombe lab

RNAi in C. elegansFire and Mello

dsRNA, DicerFlys -Zamore

Discovery ofLet-7, Ruvkin

Large numbers of miRNABartel, Tuschl, Ambros

RNAi in mammalian cells, Tuschl lab

miRNA as oncogenein vivo, Hammond lab

Direct miRNA-targetin cancer, Slack lab

AntagomirsStoffel lab

miRNA as Dx, ProgDxTakamizawa

50% miRNA in cancer regions, Calin

Transcriptionalactivation, Li et al.

miRNA as serumbiomarker, Tewari

miRNA as cancer therapy , Kota et al

1993

miRNA and SNPsShen et al.

miRNA and epigenetics

SaitomiRNA molecular

decoysEiring et al.

Cancer by singlemiRNA , Slack lab

miRNA in cancerCroce lab

Nobel Prize: Fire and Mello

FORTUNE Magazine: Biotech’s Billion Dollar

Breakthrough

Science Magazine: Breakthrough

of the Year

Sample & Assay Technologies Why quantify miRNA?


� Virtually every publication includes characterization by quantification.

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

Sample & Assay Technologies Canonical pathway of miRNA 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.

Sample & Assay Technologies Potential events that disrupt normal miRNA activity

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Disruption of miRNA–mRNA interaction

Webinar 3: Profiling miRNA expression in cells, FFPE, and serum

Altered transcriptionMethylation

Histone modificationTranscription factor

Drosha processing

Genomic instabilityAmplification/deletionTranslocationInsertional mutagenesis

Loss of miRNA binding site in targetSNP or mutationAlternative splicingLoss/change of 3′-UTR

Dicer processing


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miRNA Isolation Technologies

Functionalization

Isolation

Quantification and Profiling


Sample & Assay Technologies miRNeasy Kits: Mini, Micro

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Flexible 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


Sample & Assay Technologies miRNeasy FFPE Kit

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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


Sample & Assay Technologies miRNeasy Serum/Plasma Kit

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Purification from serum, plasma, and other fluids

� Includes synthetic RNA control assay for normalization

� Minimal elution volume (14 µl)

� High-purity RNA suitable for all downstream applications� miScript RT: up to 10 µl eluate for cDNA synthesis� One RT enough for 6, 384-well plates

� Easy, robust procedures

� Automatable protocol

QIAzol Bind Wash Elute

Manual or Automated on QIACube

Plasma Serum

Clarified plasmaor serum

For purification of circulating RNA from animal and human plasma and serum


Sample & Assay Technologies miRNA in Blood

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RBC, WBC, platelets, CTC, ‘other cells’, extracellu lar?

What is blood?RBC, WBC,platelets, other cells (e.g. circulating tumor cells)

Serum (post clotting)

Plasma (no-clotting)

� High levels of nucleases present in plasma � Freely circulating RNA should be rapidly degraded� Surprisingly, stabile miRNA can be detected in serum and plasma


Sample & Assay Technologies Stable miRNA in circulation ~2007 ��

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An evolving story

1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9:654-659

2) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e36943) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442-

174524) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human

plasma, Proc. Natl. Acad. Sci 108: 5003-50085) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.

Nat Cell Biol 13:4236) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian

cells. Nucleic Acids Res. 2010 Nov 1;38(20):7248-59.

Exosomes & micro vesicles 1,2,3

miRNA

Ago

Ago-2-miRNA complexes 4

HDL mediatedmiRNA transport 5

Other ‘protective’protein 6


Sample & Assay Technologies Extracellular miRNAs are found in all body fluids

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Weber et al. (2010)

Clinical Chem. 56: 1733-1741.

miRNeasy, miScript PCR System


Plasma Tears UrineBreastmilk

Seminalfluid

SalivaAmniotic

fluidBronchial

lavageCerebrospinal

fluidPleural

fluidPeritoneal

fluid

miR-335* miR-335* miR-515-3p miR-335* miR-518e miR-335* miR-518e miR-515-3p miR-335* miR-515-3p miR-515-3p

miR-325 miR-515-3p miR-335* miR-26a-2* miR-590-3p miR-892a miR-335* miR-335* miR-515-3p miR-892a miR-892a

miR-377* miR-137 miR-892a miR-181d miR-588 miR-515-3p miR-302c miR-509-5p miR-892a miR-223* miR-518e

miR-586 miR-509-5p miR-509-5p miR-509-5p miR-873 miR-545* miR-515-3p miR-616* miR-873 miR-616* miR-134

miR-518e miR-590-3p miR-223* miR-524-5p miR-590-5p miR-27a miR-452 miR-545* miR-509-5p miR-873 miR-509-5p

let-7i miR-873 miR-873 miR-137 miR-197 miR-518e miR-892a miR-302d miR-223* miR-335* miR-223*

miR-539 miR-892a miR-302d miR-26a-1* miR-218 miR-509-5p miR-671-5p miR-223* miR-302d miR-302d miR-515-5p

miR-616* miR-223* miR-616* miR-595 miR-515-5p miR-492 miR-515-5p miR-25* miR-616* miR-134 miR-616*

miR-302d miR-302d miR-134 miR-580 miR-137 miR-483-5p miR-137 miR-134 miR-483-5p miR-509-5p miR-302d

miR-589 miR-590-5p miR-923 miR-130a miR-335* miR-923 miR-593* miR-483-5p miR-134 miR-377* miR-873

miR-556-3p miR-195 miR-483-5p miR-515-3p miR-617 miR-873 miR-590-3p miR-923 miR-518e miR-589 miR-483-5p

miR-151-3p miR-130a miR-101* miR-513c miR-410 miR-92b miR-873 miR-518e miR-923 miR-518e miR-923

miR-548b-3p miR-675 miR-325 miR-671-5p miR-524-5p miR-302d miR-410 miR-548b-3p miR-377* miR-556-3p miR-374a

miR-192 miR-29b miR-589 miR-490-5p miR-20b let-7a miR-509-5p miR-1225-3p miR-589 miR-652 miR-598

miR-151-5p miR-616* miR-556-3p miR-367 miR-181d miR-580 miR-548d-5p miR-325 miR-556-3p miR-590-5p miR-548b-3p

miR-598 miR-410 miR-545* miR-181b miR-1 miR-616* miR-223* miR-589 miR-652 miR-923 miR-1238

mir-187 miR-101* miR-377* miR-598 miR-671-5p miR-134 miR-616* miR-92b miR-498 miR-371-3p miR-92b

miR-873 miR-134 let-7i miR-515-5p miR-509-5p miR-25* miR-148b* miR-371-3p miR-767-3p miR-890 miR-498

miR-218 miR-487b miR-890 miR-578 miR-515-3p let-7i miR-590-5p miR-324-3p miR-505 miR-498 miR-937

miR-923 miR-483-5p miR-505 miR-487b miR-892a miR-410 miR-302d miR-885-5p miR-151-5p miR-483-5p miR-377*

Sample & Assay Technologies miRNA in cancer diagnosis


Paranjape et al. (2009)

Gut. 58: 1546-1554.

Utilizing a signature of altered miRNA expression to

differentiate cancer tissue from normal tissue

Profiling circulating blood or tumor derived exosomalmiRNAs, surpassing the

invasive procedures to aid in early detection of cancers

Study SNPs in the miRNA genes, miRNA binding sites in the target

mRNA genes or in the miRNA processing/machinery pathway genes

to predict cancer predisposition

Use of miRNA based classifier to identify tissue

of origin for cancers of unknown primaries

Distinguishing tumor subtypes using a panel of

miRNAs that show differential expression within one cancer type

miRNAs in Cancer Diagnosis


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miRNA Quantification Technologies

Functionalization

Isolation



Sample & Assay Technologies miScript PCR System

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Complete miRNA quantification system

1. miScript II RT Kit� HiFlex Buffer: Unparalleled flexibility for quantify miRNA

and mRNA quantification from a single cDNA preparation� HiSpec Buffer: Unmatched specificity for mature

miRNA profiling

2. miScript miRNA PCR Arrays� miRNome� Pathway-focused

3. miScript PreAMP Kit� Optional step for small or precious samples� Full miRNome profiling from as little as 1 ng RNA

4. Assays� miScript Primer Assays� miScript Precursor Assays� QuantiTect Primer Assays

5. miScript SYBR Green PCR Kit� QuantiTect SYBR Green PCR MM� Universal Primer

6. miScript miRNA PCR Array data analysis software � Straightforward, free data analysis


Sample & Assay Technologies miScript II RT Kit

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One box with a complete miRNA quantification soluti on

miScript II RT Kit

Biogenesis studies?Mature miRNA

quantification and profiling?

HiFlex Buffer HiSpec Buffer

Flexible detection of all RNA molecules

Patent-pending technology for the specific detection of mature miRNAs

Note: Only HiSpec Buffer is recommended for use with miScript miRNA PCR Arrays


Sample & Assay Technologies miScript II RT Kit

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Reverse transcription using HiSpec Buffer

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15

20

25

30

35

40

-2 -1 0 1 2 3 4

Log (ng) of RNA in cDNA synthesis using HiSpec Buffer

Mean C

T

miR-16

miR-20a

miR-21

Linearity: 2 µg to 10 pg

12

16

20

24

28

32

1 2 3 4 5 6

Log copy number of miRNA using HiSpec Buffer

Mean C

T

miR-21

Sensitivity: 1x10 6 copies to 10 copies



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miRNA Profiling Technologies

Functionalization

Isolation



Sample & Assay Technologies miRNA expression profiling applications

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� Mechanisms of gene regulation

� Developmental biology

� Novel miRNA discovery

� Studying miRNA–mRNA and miRNA–protein interactions

� Integrative analyses of miRNAs in the context of gene regulatory networks

� Biomarkers

� Tissue-based miRNA biomarkers

� Tissues of unknown origin

� Circulating biomarkers

� Forensics

From Pritchard, C.C., et al, Nature Rev. Genet 2012, 13, 358-369


Sample & Assay Technologies miScript miRNA PCR Arrays

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Pre-formatted solutions for miRNA profiling experim ents

Pre-formatted, single use PCR arrays with wet-lab v erified assays

Fully customizable (assays and species)


� miRNome Arrays

� Most species

� Largest content

� High Content (HC) Arrays

� Targeted miRNome profiling

� Focused Arrays

� Biological relevant

� Intelligently designed

Sample & Assay Technologies miRNome miScript miRNA PCR Arrays


Leading coverage and validated assays

� Human� Mouse� Rat � Dog� Rhesus macaque

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

� Leading miRNome coverage

� Customizable

� miRBase V17 and V18 assays are available!

� Contact us if you are interest in a different species!

miRNome Arrays Benefits of miRNome Arrays

Species Assays(miRBase V16)

Human 1066

Mouse 940

Rat 653

Dog 277

Rhesus macaque 469 (V18)

Sample & Assay Technologies Focused miScript miRNA PCR Arrays


Biologically relevant, intelligently designed

Focused Arrays

� miFinder� Cancer PathwayFinder� Brain Cancer � Breast Cancer� Ovarian Cancer� Liver miFinder – New!� Apoptosis� Cell Differentiation & Development� Immunopathology � Inflammatory Response & Autoimmunity� Diabetes � Neurological Development & Disease � T-Cell & B-Cell Activation� Prostate Cancer� Cardiovascular Disease� Serum & Plasma

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

� Biological relevant and up-to-date

� Customizable

� Contact us if you are interest in a different species!

Benefits of Focused Arrays

Sample & Assay Technologies High Content (HC) miScript miRNA PCR Arrays


Targeted miRNome profiling

� miFinder 384HC

� Serum & Plasma 384HC

� Cancer PathwayFinder 384HC

� Liver miFinder 384HC – New!

Sample & Assay Technologies Anatomy of a miScript miRNA PCR Array

26

96-well Format: 84 miRNA + 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


Sample & Assay Technologies miScript PCR Controls

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Stable 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

5

10

15

20

25

30

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Brain Kidney Liver Lung Skeletal

Muscle

Testis Thymus

Mean C

T

Tissue Expression

Amplification Efficiencies

Primer 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



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Formats built to fit your cycler AND your experimen t

96-well 384-well

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



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Compatible 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



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QIAGEN PCR Array Service Core

miScript miRNA PCR Arrays

� 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!



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Rapid Workflow = Robust, Reproducible Performance

miScript miRNA PCR Arrays

1st Time Array User

� Total HeLa S3 (miRNeasy)� Pellet 1: Frozen June 2010� Pellet 2: Frozen April 2011

� HiSpec Buffered cDNA� miScript real-time PCR

� miFinder miScript miRNA PCR Array

� 1 hour� HiSpec Buffer

� 2 minutes

� 2 hours

� 15 minutes



32

An entire miRNome from only 10 ng of total RNA

miScript PreAMP PCR Kit: decrease input up to 1000X


� Universal reference RNA� Two operators

� Experienced operator� First time operator

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

Operator Variability Assessment

Sample & Assay Technologies miRNome miRNA expression profiling

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HCT 116 colorectal cancer cells

Scatter Plot Volcano Plot

1.E-05

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1.E+02

1.E-05

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1.E+02

2-∆CT

: Untreated

2-∆CT: Treated

� 5-aza-2’-dC irreversibly inhibits DNA methyltransferase driven DNA methylation reactions by incorporating into DNA and covalently binding to the active site of the DNMT.

� Scatter Plot: Significant differences exist in the mature miRNA expression levels of the two samples that were tested

� 104 miRNAs were strongly up-regulated in 5-aza-2’-dC treated cells, while 30 were strongly down-regulated in 5-aza-2’-dC treated HCT 116 cells.

� Volcano Plot: When a p Value of 0.05 is applied, the expression up-regulation of 89 of the 104 miRNAs is significant, and the expression down-regulation of 21 of the 30 miRNAs is significant

0.00001

0.0001

0.001

0.01

0.1

1

-8 -6 -4 -2 0 2 4 6 8

Log2 (Fold-Regulation)

p Value


Sample & Assay Technologies Focused miRNA expression profiling

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FFPE samples


Reproducibility 2-∆CT: Lung Tumor vs. Normal

� One 5 µm FFPE section used per FFPE isolation� On average, each isolation provided enough total RNA for:

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

� RT: 125 ng total RNA, HiSpec Buffer� qPCR: Human miFinder miScript miRNA PCR Array (0.5 ng cDNA per well)� Significant differences exist between the miRNA expression levels of the two tissue types

Sample & Assay Technologies miScript PreAMP: Excellent result with 250-fold less input

35

Limiting FFPE sample

� 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 t han 2 full human miScript

miRNome profiles. Input was only 1 ng of cDNA!



36

miRNA Expression Profiling from Body Fluids


-80°C vs. RT for 24 hrs Freeze-thaw cycles Plasma vs. Serum


37

Workflow: Serum & plasma miRNA expression profiling

1. Collect whole blood and separate serum or plasma

2. Recommended starting amount of serum or plasma: 100–200 µl� Note: If starting with 50 µl (or less) of serum or plasma, incorporate preamplification

3. Isolate RNA: miRNeasy Serum/Plasma Kit + miRNeasy Serum/Plasma Spike-in Control

4. Reverse transcription: Perform miScript II RT reaction (using HiSpec Buffer)� Per intended 384-well plate , reverse transcribe 1.5 µl RNA eluate (1/10th prep)

5. (Optional) Preamplification: If the starting amount of serum or plasma is 50 µl (or less), perform preamplification using the miScript PreAMP PCR Kit

6. Real-time PCR: Profile miRNA expression using chosen miScript miRNA PCR Array� Array recommendations:

– miRNome miScript miRNA PCR Array– Serum & Plasma 384HC miScript miRNA PCR Array– Serum & Plasma miScript miRNA PCR Array


Sample & Assay Technologies miRNA expression profiling using serum samples

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High Content (HC) Array: 384 assays

2-∆CT: Cancer vs. Normal

� Workflow� 200 µl serum � 14 µl total RNA � 1.5 µl total RNA, HiSpec Buffer � Serum & Plasma 384HC miScript

miRNA PCR Array

� Significant differences exist between the miRNA expression levels of the two serum types� ± 3-fold [red lines] used as a cutoff for significance


Reproducibility

Sample & Assay Technologies miScript PreAMP: Excellent result with 300-fold less input

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Limiting serum sample

� Workflow� 5 µl serum � 14 µl total RNA � 2 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

Reproducibility 2-∆CT: Colorectal Cancer vs. Normal



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Workflow: Urine miRNA expression profiling

1. Recommended starting amount of urine: 200 µl

2. Isolate RNA: miRNeasy Serum/Plasma Kit + miRNeasy Serum/Plasma Spike-in Control

3. Reverse transcription: Reverse transcribe 1.5 µl RNA eluate (1/10th prep) using a HiSpec buffered miScript II RT Kit reaction

4. Preamplification: Preamplify cDNA using the miScript PreAMP PCR Kit and themiScript PreAMP Primer Mix of interest

5. Real-time PCR: Using amplified cDNA, profile miRNA using chosen miScript miRNA PCR Array� Array recommendations:

– miRNome miScript miRNA PCR Array– miFinder 384HC miScript miRNA PCR Array– miFinder miScript miRNA PCR Array


Preamplification is highly recommended

No PreAMP: High C T values

PreAMP: Robust C T values

Sample & Assay Technologies Urine miRNA profiling: PreAMP is recommended

41

Results are highly reproducible


� Workflow� 200 µl urine � 14 µl total RNA � 1.5 µl total RNA, HiSpec Buffer � one-tenth of cDNA, miScript

PreAMP � preamplified cDNA used for Human miFinder miScript miRNA PCR Array

� Conclusion: Starting with only 200 µl urine, preamplification enables accurate, reproducible miRNA expression profiling


42

Biomarker discovery workflow


Concept

Phase I (determined expressed miRNAs):Pooled sample profiling,

Maximal Assays (miRNome or 384HC)

Phase II (determine differentially expressed miRNAs ):Individual profiling of samples (that went into poo ls),

Only Expressed miRNAs (< 384-well plate)

Phase III (statistical power):Individual profiling of all samples in study,

Differentially Expressed miRNAs from Phase II (mult iple samples per 384-well plate)


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Biofluids: miRNA data normalization


Sample & Assay Technologies Data normalization

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Check reverse transcription (miRTC) and PCR (PPC) c ontrols


Position Control C T: Serum Sample A C T: Serum Sample B

H09 miRTC 18.8 18.5

H10 miRTC 18.7 18.6

H11 PPC 19.4 19.6

H12 PPC 19.6 19.8

� As determined by the raw CT values, the reverse transcription and PCR efficiency of both samples are highly comparable

� CT values differ by less than 0.25 units

Step 1


45

Check snoRNAs/snRNA controls


Step 2

� snoRNAs/snRNAs exhibit borderline detectable expression� As is often found with serum samples, standard housekeeping

assays cannot be used for data normalization� In fact, this data indicated a prep free of cellular contamination

Control C T: Serum Sample A C T: Serum Sample B

SNORD61 36.3 34.3

SNORD68 34.6 35.0

SNORD72 35.0 35.0

SNORD95 31.1 39.3

SNORD96A 33.6 34.5

RNU6-2 37.9 39.1

Sample & Assay Technologies miScript PCR Controls

46Webinar 3: Profiling miRNA expression in cells, FFPE, and serum

Strong expression in 100% blood, low expression in serum

Target100% Blood:

CT Mean100% Serum:

CT Mean∆CT

(Serum - Blood)

RNU6-2 21.9 34.4 12.5

SNORD61 24.6 35.4 10.8

SNORD68 20.7 33.4 12.7

SNORD96A 23.1 34.1 11.0

SNORD95 21.6 30.9 9.3

SNORD72 27.0 35.1 8.1

If the mean C T of the miScript PCR Control panel is lower than 32, then you have greater than 0.1% cellular contamination f rom cells

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20

24

28

32

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100 50 10 1 0.1 0.01 0.001 0.0001

Blood Percentage (%)

Mean C

T

cel-miR-39

miScript Controls

33.7419.030.0001

33.9419.670.001

33.7519.420.01

33.5219.910.1

30.0318.701

26.3718.0810

24.4118.9250

23.5219.24100

CT Mean:miScript Controls

CT Mean:cel-miR-39

Blood Percentage(%)

33.7419.030.0001

33.9419.670.001

33.7519.420.01

33.5219.910.1

30.0318.701

26.3718.0810

24.4118.9250

23.5219.24100


CT Mean:cel-miR-39

Blood Percentage(%)

If the mean C T of the miScript PCR Control panel is lower than 32, then you have greater than 0.1% cellular contamination f rom cells

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20

24

28

32

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100 50 10 1 0.1 0.01 0.001 0.0001


Mean C

T

cel-miR-39

miScript Controls

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20

24

28

32

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100 50 10 1 0.1 0.01 0.001 0.0001


Mean C

T

cel-miR-39

miScript Controls

33.7419.030.0001

33.9419.670.001

33.7519.420.01

33.5219.910.1

30.0318.701

26.3718.0810

24.4118.9250

23.5219.24100


CT Mean:cel-miR-39

Blood Percentage(%)

33.7419.030.0001

33.9419.670.001

33.7519.420.01

33.5219.910.1

30.0318.701

26.3718.0810

24.4118.9250

23.5219.24100


CT Mean:cel-miR-39

Blood Percentage(%)


47Webinar 3: Profiling miRNA expression in cells, FFPE, and serum

Step 3

� Calibrate data of each plate to its RNA Recovery Co ntrols�Can only be used if Syn-cel-miR-39 miScript miRNA Mimic is spiked into the

sample during RNA preparation

Then: If your array is ‘unbiased’ (full miScript PC R Array)

� Normalize to the C T mean of all expressed targets on plate (OK)

OR

� Normalize to the C T mean of commonly expressed targets on plate (better)Or: If your array is ‘biased’ (small panel of regul ated miRNAs)

� Normalize data to C T of several invariant miRNAs

Sample & Assay Technologies Where can I find miScript miRNA PCR Arrays?

48

www.sabiosciences.com/mirna_pcr_array.php

� miRNA Overview

� miScript PCR System

� miScript miRNA PCR Arrays

� Products for functional studies

� miRNA purification options


Sample & Assay Technologies Where can I find miScript Primer Assays?

49

www.qiagen.com/GeneGlobe


Sample & Assay Technologies RT2 Profiler miRNA Targets PCR Array

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What genes are regulated by your miRNA of interest?

Benefits:� Assist miRNA functional analysis� Follow up on miRNA expression profiling analyses� Species currently covered: Human, mouse, and rat

Features:� Profiles expression of 84 verified & bioinformatically predicted target genes� Target genes specific for a miRNA & others in same seed sequence family� Free data analysis suite� Available in 2, 12 or 24 plate packs� Compatible with any real time qPCR instrument currently in your lab

Note: Uses RT 2 qPCR Reagent Kits



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miRNeasy Mini Kit, miRNeasy Micro Kit miScript II RT K it and PreAMP Kit HiPerFect Transfection Reagent

miRNeasy 96 Kit miScript SYBR Green PCR Kit Attractene Transfection R eagent

miRNeasy FFPE Kit miScript miRNA PCR Arrays miScript miRNA Mimics

miRNeasy Serum/Plasma Kit miScript miRNA Data Analysi s Tool miScript miRNA Inhibitors

PAXgene Tissue miRNA Kit miScript Primer Assay Custom miScript miRNA Mimics

PAXgene Blood miRNA Kit miScript Precursor Assay miSc ript Target Protector

Supplementary protocol for miRNA from Plasma and Serum

miScript PCR Starter KitmiScript miRNA Inhibitor 96 and 384 Plates and Sets

Pro

filin

g

QIAcube QIAgility Rotor-Gene Q


QIAGEN Service Core

FunctionalizationIsolationQuantification and profiling

QIAGEN’s miRNA portfolio

Your miRNA workflow, from sample to results!

Sample & Assay Technologies Upcoming webinars


May 2013

Sign up at http://www.SABiosciences.com/seminarlist .php

Sample & Assay Technologies Thank you for attending

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http://www.sabiosciences.com/promotion/miscriptdemo.php

Technical Support Contact Information

� Monday through Friday

� 8:00AM to 6:00PM EST

� Direct Phone: 888-503-3187

� E-Mail: [email protected]


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Thank you for attending today’s webinar!

Jonathan Shaffer, Ph.D.

[email protected] Technologies, R&D Americas

Questions?


Documents

Mi rna part iii_2013