mi rna assays 2012

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Sample & Assay Technologies From Cellular to Molecular – New Assays for miRNA Research Samuel J. Rulli, Jr. Ph.D. [email protected]

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Page 1: Mi rna assays 2012

Sample & Assay Technologies

From Cellular to Molecular –New Assays for miRNA Research

Samuel J. Rulli, Jr. [email protected]

Page 2: Mi rna assays 2012

Sample & Assay Technologies

From Cellular to Molecular –New Assays for miRNA Research

Samuel J. Rulli, Jr. [email protected]

Questions, Comments, Concerns?US Applications Support

[email protected]

Questions, Comments, Concerns?Global Applications Support

[email protected]

Page 3: Mi rna assays 2012

Sample & Assay Technologies- 3 -

miRNA Analysis: From Cellular to Molecular

SureFIND Transcriptome PCR Arrays

Target Identification Expression

Isolation

Function

miScript miRNA PCR Arrays

miRNeasy

miRNAStudies

miRNA mimics & inhibitorsCignal Reporters

Page 4: Mi rna assays 2012

Sample & Assay Technologies- 4 -

What Is Micro RNA?

Endogenously expressed small functional RNAs (~ 21nt -24 nt)

Regulate mRNA expression post-transcriptionally Involves the formation of RISC (RNA induced Silencing Complex) Translational Repression and or mRNA degradation

Only partial sequence complementarity required A given miRNA can regulate multiple mRNAs A given mRNA can be regulated by multiple miRNAs Another layer of complexity for regulating gene expression

Aberrant expression of miRNAs is associated with many diseasese.g. Many different Cancers

Ω

Seed region

5’ 3’

miRNA

Target mRNA

5’3’CDS

RISC

Ω

Seed region

5’ 3’

miRNA

Target mRNA

5’3’CDS

RISC

Ω

Seed region

5’ 3’

miRNA

Target mRNA

5’3’CDS

RISC

Page 5: Mi rna assays 2012

Sample & Assay Technologies- 5 -

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

Page 6: Mi rna assays 2012

Sample & Assay Technologies- 6 -

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

Page 7: Mi rna assays 2012

Sample & Assay Technologies- 7 -

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

Page 8: Mi rna assays 2012

Sample & Assay Technologies- 8 -

A growing picture of miRNA dysregulation in cancer pathways

.In the last 5 years, a substantial number of studies have associated

.various miRNAs with cell proliferation, resistance to apoptosis, differentiation, immunity and cancer initiation, progression and metastasis.

.For example:

Deletions of miRNA-regulated genes have been detected in more than 65% of chronic lymphocytic leukemia cases, in 50% of mantle-cell lymphomas, in 16% to 40% of multiple myelomas, and in 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

Page 9: Mi rna assays 2012

Sample & Assay Technologies- 9 -

Why Analyze miRNA?

.Easy to profile entire miRNome with only ~1000 miRNAs

.Complexity of miRNA/mRNA interaction makes them exciting and unknown epigenetic mechanism of gene regulation

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

Cancer Development Immunology Aging Heart Diseases Neurological Diseases Diabetes

miRNAs are promising biomarkers for early disease d etection and prognosis

Page 10: Mi rna assays 2012

Sample & Assay Technologies- 10 -

SureFIND Transcriptome PCR Arrays

Target Identification Expression

Isolation

Function

miRNeasy

miRNAStudies

miRNA mimics & inhibitorsCignal Reporters

miRNA Analysis: From Cellular to Molecular

miScript miRNA PCR Arraysand PCR Assays

Page 11: Mi rna assays 2012

Sample & Assay Technologies- 11 -

miRNeasyPurification of miRNAs

miRNA purificationCells and tissues miRNeasy Mini Kit

Cells and tissues (small amounts) miRNeasy Micro Kit

Cells and tissues, 96-well format miRNeasy 96 Kit

Formalin-fixed, paraffin-embedded tissues miRNeasy FFPE Kit

Plasma and serum miRNeasy Serum/Plasma Kit

Almost any sample source can yield high quality miRNA for miRNA profiling

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Sample & Assay Technologies- 12 -

miRNA isolation is simple from any sample

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Sample & Assay Technologies- 13 -

miRNA isolation is simple from any sample

Sufficient for miScriptmiRNA PCR Arrays

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Sample & Assay Technologies- 14 -

miRNA isolation is simple from any sample

Maximize limited sample amounts to measure miRNAand mRNA by PCR arrays or individual qPCR Assays

Page 15: Mi rna assays 2012

Sample & Assay Technologies- 15 -

SureFIND Transcriptome PCR Arrays

Target Identification Expression

Isolation

Function

miRNeasy

miRNAStudies

miRNA mimics & inhibitorsCignal Reporters

miRNA Analysis: From Cellular to Molecular

miScript miRNA PCR Arraysand PCR Assays

Page 16: Mi rna assays 2012

Sample & Assay Technologies- 16 -

How to Monitor miRNA Expression Levels

.Northern Blot Very low throughput Very time consuming and complex Specificity issue

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

.Real-Time RT-PCR (qPCR) Simpler Protocol Better Sensitivity, Specificity & Reproducibility No investment in equipment Profile up to 384 miRNA in a single run (miRNome needs 3x 384 plates)

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Sample & Assay Technologies- 17 -

miScript miRNA PCR Array System

.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: Search miRNA Primers:

http://www.sabiosciences.com/mirna_pcr_assay.php

PCR Arrays: Pathways and Genomes Cancer Cell Differentiation & Development Immunopathology Inflammation miFinder Whole Genome (miRNome) – Sanger miRBase V14.0 Serum (or plasma) Brain Cancer miRNA Neurological Development & Disease

.Optimized miScript SYBR® Green Master Mix

.miScript miRNA Data Analysis Excel Template & Web Portal

-or-

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Sample & Assay Technologies- 18 -

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

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Sample & Assay Technologies- 19 -

Universal miRNA RT-PCR used by miScript miRNA PCR Arrays and Assays

TTTTTTTT

miRNA

AAAAAAAA

NNTTTTTTTTUniversal RT Primer site

Reverse Transcriptase

Poly(A) Polymerase

Real-Time PCR

TTTTTTTT

miRNA-Specific Primer

Universal Primer

Same cDNA preparation can assay ANY miRNA now or in the future (think miRBase 24)

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Sample & Assay Technologies- 20 -

Performance: Universal RT Advantages

.Benefit of universal RT 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

Similar potential specificity, & additional flexibility for future discveries

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Sample & Assay Technologies- 21 -

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

Page 22: Mi rna assays 2012

Sample & Assay Technologies- 22 -

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

Page 23: Mi rna assays 2012

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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

Page 24: Mi rna assays 2012

Sample & Assay Technologies- 24 -

miScript miRNA PCR ArraysRedefining miRNA expression profiling

Human, Mouse, Rat: miFinder (Dog and Rhesus macaque) miFinder 384 HC (high content)

Cancer PathwayFinder Brain Cancer Breast Cancer Cancer PathwayFinder Ovarian Cancer Prostate Cancer

Immunopathology T cell & B cell Activation Inflammatory Response & Autoimmunity Neurological Development & Disease

Cell Differentiation & Development (Stem Cell)

Serum & Plasma

Fully customizable!

Complete miRNome Arrays Pathway and Disease -Focused Arrays

Human Mouse Rat Dog New! Rhesus macaque

Species Assays(miRBase V16)

Human 1066

Mouse 940

Rat 653

Dog 277

Rhesus macaque 469 (V18)

Page 25: Mi rna assays 2012

Sample & Assay Technologies- 25 -

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

Page 26: Mi rna assays 2012

Sample & Assay Technologies- 26 -

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

Page 27: Mi rna assays 2012

Sample & Assay Technologies- 27 -

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

Page 28: Mi rna assays 2012

Sample & Assay Technologies- 28 -

Web Portal Location

http://pcrdataanalysis.sabiosciences.com/mirna/arrayanalysis.php

Page 29: Mi rna assays 2012

Sample & Assay Technologies- 29 -

Average ∆Ct Fold Change Fold Regulation

Analyze Results

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Sample & Assay Technologies- 30 -

Heat Map

Visualization of Data

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Sample & Assay Technologies- 31 -

Visualization of Data

Scatter Plot

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Sample & Assay Technologies- 32 -

Visualization of Data

Volcano Plot

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Sample & Assay Technologies- 33 -

Clustergram Clustergram

Visualization of Data

Page 34: Mi rna assays 2012

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Application Examples

CancerIdentify miRNA biomarkers

Development & DifferentiationRegulation of Cell Type by miRNAs

Genome-Wide DiscoveryProfile downstream miRNA expression

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Application Data: Which Cancer-related miRNAs are expressed in a spec ific tumor?

Human miScript CancerFinder miRNA PCR Array:Identified miRNA dysregulated in a Colon Tumor.

Page 36: Mi rna assays 2012

Sample & Assay Technologies- 36 -

Application Data: Cell Differentiation & Developmen t

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

26 Brain-Specific miRNA

20 Muscle-Specific miRNA

Page 37: Mi rna assays 2012

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Application Data: Genome-Wide Screening

Human miScript miRNome PCR Array: Identifies Known and Novel miRNA Targets of the p53 Signaling Pathway

15

20

25

30

35

40

15 20 25 30 35 40Ct Control

Ct A

den

o-p5

3

34a 940

203 551a

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Sample & Assay Technologies- 38 -

4

8

12

16

20

24

28

32

36

40

1 7 13 19 25 31 37 43 49 55 61 67 73

CT Value

FFPE Isolation 1

FFPE Isolation 2

FFPE Isolation 3

4

8

12

16

20

24

28

32

36

40

1 7 13 19 25 31 37 43 49 55 61 67 73

Human miFinder miRNA Assay

CT Value

FFPE Isolation 1

FFPE Isolation 2

FFPE Isolation 3

0

2

4

6

8

10

12

14

16

18

20

N: 1

-1

N: 1

-3

N: 2

-2

T: 1-2

T: 2-1

T: 2-3

T: 3-2

T: 4-1

T: 4-3

T: 5-2

T: 6-1

T: 6-3

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

Page 39: Mi rna assays 2012

Sample & Assay Technologies- 39 -

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

2-∆CT

: Normal Lung FFPE Tissue

2-∆CT: Tumor Lung FFPE Tissue

miRNA expression profiling using FFPE samples (cont .)

-14

-10

-6

-2

2

6

10

14

Log2 Fold-Regulation (Tumor vs. Norm

al)

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 arbitrary cutoff

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Sample & Assay Technologies- 40 -

1.E-05

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1.E-05

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

2-∆CT

: Normal Serum2-∆CT: Breast Cancer Serum

miR-34a

14

18

22

26

30

34

38

1 6 11 16 21 26 31 36 41 46 51 56 61

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

Page 41: Mi rna assays 2012

Sample & Assay Technologies- 41 -

SureFIND Transcriptome PCR Arrays

Target Identification Expression

Isolation

Function

miRNA mimics & inhibitorsCignal Reporters

miRNeasy

miRNAStudies

miRNA Analysis: From Cellular to Molecular

miScript miRNA PCR Arraysand PCR Assays

Page 42: Mi rna assays 2012

Sample & Assay Technologies- 42 -

Cignal Reporter System: Measure Pathway Activity in Cells

Cignal Reporter Assays enable researchers to monitor transcription factor activation

Flexible system allows monitoring of different reporter assays (GFP or Luciferase)

Available as single assays or grouped by biology or pathway

Validated assays for high signal to noise ratios

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Sample & Assay Technologies- 43 -

What Cignal Reporter Assays Provide

Functionally validated dual-reporter formulation

. Minimizes variability, increasing the biological relevance of each experiment

Ready-to-use reporters and controls, coupled with a transient reporter system

Enables simple and rapid analysis of the regulation of 45 signal transduction pathways

Combination of custom-engineered transcriptional regulatory elements (TREs) and destabilized, codon-optimized firefly luciferase

Increases the signal to noise ratio and maximizes assay sensitivity and specificity

Page 44: Mi rna assays 2012

Sample & Assay Technologies- 44 -

Reporter Construct firefly luciferaseTATAbox

Tandem repeats of TRE

Pathway-targeted Transcriptional Regulatory Element s (TRE), which establish the

pathway specificity of each reporter

Cignal Reporter Assays: Complete SolutionHow Cignal Reporter Assays Work

TF FL

Upstream SignalingEvents

Page 45: Mi rna assays 2012

Sample & Assay Technologies- 45 -

Reporter Design: Dual-Luciferase Assays

Reporter Construct Firefly luciferaseTATAboxTRE

Renilla luciferaseTATAboxCMVNormalization Construct

Pathway-targeted transcriptionalregulatory elements (45)

Constitutive transcriptionalregulatory element (1)

The Transcriptional Regulatory Elements (TREs) are what establish the

pathway specificity of each reporter!

Page 46: Mi rna assays 2012

Sample & Assay Technologies- 46 -

Cignal Reporter Assays (45 Pathways)

Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GliHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR

Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2c-myc Myc/Max Nanog NanogNFκB NFκBNotch RBP-Jk Oct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARProgesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4 VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR

Page 47: Mi rna assays 2012

Sample & Assay Technologies- 47 -

Cignal Pathway Reporter System Products

Easy totransfect cell lines

Primary cells, stem cells,and difficult to transfect cell lines

EndpointAssays

EndpointAssays

Dynamic Live Cell Assays

Dynamic Live Cell Assays

Cignal Dual-Luciferase Reporter Assays

Cignal Lenti Luciferase Reporters

Cignal GFP Reporter Assays

Cignal Lenti GFP Reporters

Single PathwayAssays

Single PathwayAssays

Single PathwayAssays

Single PathwayAssays

10-PathwayArrays

10-PathwayArrays

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Sample & Assay Technologies- 48 -

Cignal Reporters: What You Need

Cignal Reporter Assay

Reliable transfection reagent and protocol (Attractene, HiPerFect)

Biological TestmiRNA mimics & Inhibitors

Detection Instrument and Luciferase Substrates

Page 49: Mi rna assays 2012

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Solutions for microRNA Function

QIAGEN miRNA Mimics and InhibitorsFor functional analysis of miRNA

QIAGEN Target ProtectorsDisrupt Specific miRNA-Target Interactions

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Sample & Assay Technologies- 50 -

miRNA Mimics & Inhibitors: How do they work?

Transfection of Mimics

Transfection of Inhibitors

RISC

Target Expression Level

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Sample & Assay Technologies- 51 -

miRNA Mimics & Inhibitors: How do they work?

Transfection of Mimics

Transfection of Inhibitors

RISC

Target Expression Level

Mimics increase miRNA activity

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Sample & Assay Technologies- 52 -

miRNA Mimics & Inhibitors: How do they work?

Transfection of Mimics

Transfection of Inhibitors

RISC

Target Expression Level

Inhibitor decrease miRNA activity

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Application Example: miRNA & Pathway Activity

.The let-7 family of miRNAs is widely viewed as playing a role in tumor suppression.

.Expression of let-7 family members is downregulated in many cancer types and during tumor progression in comparison to normal tissue.

.Humans have 10 mature let-7 family sequences (let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, miR-98, and miR-202)

.Interestingly, certain let-7 species are also known to be upregulated in some cancer types. These data on the expression of let-7 in various cancers indicate that individual let-7 family members can have different activities.

.Question: Which let-7 family members effect cell cy cle progression and how?

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Sample & Assay Technologies- 54 -

Cignal Reporters: What You Need

• Cignal Cell Cycle Pathway ReporterMeasures E2F/DP1 Activity

• Transfection of HEK293 cells

• Biological Test •Let-7 family mimics

• Dual-Luciferase Assay

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Sample & Assay Technologies- 55 -

Let-7 Family Members and the Cell Cycle

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Sample & Assay Technologies- 56 -

SureFIND Transcriptome PCR Arrays

Target Identification Expression

Isolation

Function

miRNeasy

miRNAStudies

miRNA mimics & inhibitorsCignal Reporters

miRNA Analysis: From Cellular to Molecular

miScript miRNA PCR Arraysand PCR Assays

Page 57: Mi rna assays 2012

Sample & Assay Technologies

Discover miRNAs Regulating the Expression of any Gene

Page 58: Mi rna assays 2012

Sample & Assay Technologies- 58 -

GOI

What regulates your gene?

mRNA

DNA

miRNA

TranscriptionalFactors

Upstream Pathway Regulators

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Sample & Assay Technologies- 59 -

How do you identify those regulators?

.Overexpression

.RNAi knockdown

.Chemical Treatments.Promoter Analysis

.Forward Genetics

.Bioinformatics

mRNA

DNA

×

×

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Sample & Assay Technologies- 60 -

miRNA mimic

Approach to Identifying miRNA Regulators

miRNA

1) Treat Cells

2) Isolate RNA

3) First Strand cDNA Synthesis

4) qPCR for Gene Expression

1 2 3 4

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Sample & Assay Technologies- 61 -

Transcriptome cDNA panels

.Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the miRNAs, Pathways or Transcription Factors that regulate the expression of any gene.

.Each Transcriptome PCR Array is a panel of Transcriptomes or cDNAs: 90 Experimental cDNA samples 4 Vehicle Control cDNA samples 2 PCR Controls (only for SYBR green reactions)

.Each experimental cDNA sample is derived from cells treated with a different treatment.

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Sample & Assay Technologies- 62 -

Example Transcriptome PCR Array

cDNA Sample 1

cDNA Sample 2

cDNA Sample 3

cDNA Sample 4

cDNA Sample 5

cDNA Sample 6

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Materials needed for miRNA discovery

.Transcriptome PCR Array (1 for each GOI or HKG)

.The Research Also Needs:

.qPCR Thermocycler

Micropipettor

qPCR Master Mix

qPCR Assay (either SYBR® green or Probe-based Assays)

Computer with Microsoft Excel for Data Analysis

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Sample & Assay Technologies- 64 -

SureFIND Protocol

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Sample & Assay Technologies- 65 -

Application Example: miRNA regulation of INPPL1

.INPPL1, also known as SHIP2, is a SH2-containing 5'-inositol phosphatase

.Converts PIP3 to PtdIns(3,4)P2, which negatively regulates PI3K signaling

.Linked to Carcinogenesis

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Sample & Assay Technologies- 66 -

Site Predictions for INPPL1

TargetScan PicTar miRANDA

miR-10 miR-184 miR-18a

miR-135 miR-205 miR-18b

miR-146 miR-342-3p

miR-184 miR-642

miR-18ab miR-671-3p

miR-205

miR-27ab

miR-29abc

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Sample & Assay Technologies- 67 -

Cancer miRNA Transcriptome PCR Array Results for INPPL1

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Sample & Assay Technologies- 68 -

Site Predictions for INPPL1

TargetScan PicTar miRANDA

miR-10 miR-184 miR-18a

miR-135 miR-205 miR-18b

miR-146 miR-342-3p

miR-184 miR-642

miR-18ab miR-671-3p

miR-205

miR-27ab

miR-29abc

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Sample & Assay Technologies- 69 -

Compare Results with Research Community

What’s really exciting….

Three new miRNAs that play a role in INPPL1 express ion

mir-193bmir-138mir-373

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Now what?............. Suggestions to Validate your “Hits”

.miRNA-based Discovery Perform a miScript miRNA mimic experiment

Identical Gain-Of-Function experiment Perform a miScript miRNA inhibitor experiment

Loss-of-Function Experiment Peform a miScript Target Protector experiment

ssRNA that disrupt a specific Target-miRNA interaction

Page 71: Mi rna assays 2012

Sample & Assay Technologies- 71 -

SureFIND Transcriptome PCR Arrays

Target Identification Expression

Isolation

Function

miScript miRNA PCR Arraysand PCR Assays

miRNeasy

miRNAStudies

miRNA mimics & inhibitorsCignal Reporters

Summary: miRNA Analysis: From Cellular to Molecular

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Sample & Assay Technologies- 72 -

Questions, Comments, Concerns or Suggestions?

Answers to your technical and biological questions:

Samuel [email protected]

Copy of slides? Type slides into questions box or fill out survey on exitWant someone to call you? Type contact me into questions boxWant to try something? Request demos in the questio ns box

miRneasy isolation; -miScript miRNA Arrays; miRNA mimics/inhibitors; target protectors; Cignal Reporter assays; etc.

Questions, Comments, Concerns?US Applications Support

[email protected]

Questions, Comments, Concerns?Global Applications Support

[email protected]

*SURVEY?