mi rna assays 2012
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Sample & Assay Technologies
From Cellular to Molecular –New Assays for miRNA Research
Samuel J. Rulli, Jr. [email protected]
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Sample & Assay Technologies
From Cellular to Molecular –New Assays for miRNA Research
Samuel J. Rulli, Jr. [email protected]
Questions, Comments, Concerns?US Applications Support
Questions, Comments, Concerns?Global Applications Support
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Sample & Assay Technologies- 23 -
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
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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)
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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
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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
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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
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Sample & Assay Technologies- 28 -
Web Portal Location
http://pcrdataanalysis.sabiosciences.com/mirna/arrayanalysis.php
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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
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Sample & Assay Technologies- 34 -
Application Examples
CancerIdentify miRNA biomarkers
Development & DifferentiationRegulation of Cell Type by miRNAs
Genome-Wide DiscoveryProfile downstream miRNA expression
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Sample & Assay Technologies- 35 -
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.
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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
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Sample & Assay Technologies- 37 -
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
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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
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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
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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
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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
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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!
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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
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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
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Sample & Assay Technologies- 49 -
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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Sample & Assay Technologies- 53 -
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
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Sample & Assay Technologies
Discover miRNAs Regulating the Expression of any Gene
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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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Sample & Assay Technologies- 63 -
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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Sample & Assay Technologies- 70 -
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
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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
Questions, Comments, Concerns?Global Applications Support
*SURVEY?