mi rna functional analysis 2013
TRANSCRIPT
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Sample & Assay Technologies- 2 -
Topics for webinar
What is microRNA?Manipulation of miRNA in cell lines using reporter assays
miRNA Mimics InhibitorsTarget protectors
Screening your target gene for miRNA regulationReady-to-use cDNA panels
Identification of miRNA regulation by qPCRmiRNA Targets PCR Arrays
SummaryQ & A session
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Sample & Assay Technologies- 3 -
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- 4 -
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- 5 -
A growing picture of miRNA dysregulation in cancer, developmental and metabolic pathways
.In the last 5 years, a substantial number of studies and reviews have associated
.the presence of various miRNAs with cell proliferation, resistance to apoptosis, and
.differentiation in cancer cells.
.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- 6 -
Topics for webinar
What is microRNA?Manipulation of miRNA in cell lines using reporter assays
miRNA Mimics InhibitorsTarget protectors
Screening your target gene for miRNA regulationReady-to-use cDNA panels
Identification of miRNA regulation by qPCRmiRNA Targets PCR Arrays
SummaryQ & A session
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Sample & Assay Technologies- 7 -
Manipulation of miRNA in cell lines
Functional interactions between miRNA and mRNA can be follow using:
Reporter assays: reporter gene has miRNA site in 3’ UTR which decreases reporter gene translation or causes transcriptional degradation
Real-time PCR: monitors transcriptional degradation of mRNA
Western blot: monitors transcriptional or translational changes
Pathway reporter assay: monitors the pathway activity of specific transcriptionFactors.
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Sample & Assay Technologies- 8 -
miRNA Mimics & Inhibitors: How do they work?
Transfection of Mimics
Transfection of Inhibitors
RISC
Target Expression Level
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Sample & Assay Technologies- 9 -
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- 10 -
Comparable down-regulation by mimics and endogenous miRNAs
Hela cells with reporter assays for miR-1 or miR-16. miR-16 is endogenously Expressed, while miR-1 was introduced using a miRNA mimic
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Sample & Assay Technologies- 11 -
Stability of miRNA mimics
miRNA mimics are stable for at least 72 hours.
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Sample & Assay Technologies
HDAC4
HDAC4 is a target of mir-1
HDAC4 had 2 miR-1 interaction sites. HeLa cells transfected with mir-1 show downRegulation of HDAC4 at the mRNA and protein levels.
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Sample & Assay Technologies- 13 -
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- 14 -
mir-16 is decreased by miR-16 inhibitor in HeLa cells by reporter assay
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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- 16 -
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- 17 -
Cignal Reporter Assays (45 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREBCell Cycle E2F/DP1DNA Damage p53EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ERGATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GLIHNF4 HNF4Hypoxia HIF-1Interferon Regulation IRF-1Type I Interferon STAT1/STAT2Interferon Gamma STAT1/STAT1KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRFMAPK/JNK AP-1 MEF2 MEF2c-myc Myc/MaxNanog NanogNFκB NFκBNotch RBP-Jk Oct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFATPPAR PPARProgesterone PRRetinoic Acid RARRetinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4 Vitamin D Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
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Sample & Assay Technologies- 18 -
Let-7 Family Members and the Cell Cycle
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Sample & Assay Technologies- 19 -
Topics for webinar
What is microRNA?Manipulation of miRNA in cell lines using reporter assays
miRNA Mimics InhibitorsTarget protectors
Screening your target gene for miRNA regulationReady-to-use cDNA panels
Identification of miRNA regulation by qPCRmiRNA Targets PCR Arrays
SummaryQ & A session
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Sample & Assay Technologies- 20 -
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- 21 -
Transcriptome cDNA panels
.Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the miRNAs, 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 miRNA mimic treatment.
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Sample & Assay Technologies- 22 -
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- 23 -
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- 24 -
SureFIND Protocol
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Sample & Assay Technologies
miRNA pathways available
• Cancer miRNAs
• Immunopathology
• Development and differentiation
• Complete miRNome (~900 miRNAs)
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Sample & Assay Technologies- 26 -
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- 27 -
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- 28 -
Cancer miRNA Transcriptome PCR Array Results for INPPL1
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Sample & Assay Technologies- 29 -
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- 30 -
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- 31 -
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- 32 -
Topics for webinar
What is microRNA?Manipulation of miRNA in cell lines using reporter assays
miRNA Mimics InhibitorsTarget protectors
Screening your target gene for miRNA regulationReady-to-use cDNA panels
Identification of miRNA regulation by qPCRmiRNA Targets PCR Arrays
SummaryQ & A session
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Sample & Assay Technologies- 33 -
Identification of transcriptional degradation using qPCR: RT2 Profiler microRNA Targets PCR Arrays
Transfection of Mimics
Transfection of Inhibitors
RISC
Target Expression Level
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Sample & Assay Technologies- 34 -
• 84 Experimentally verified or predicted targets of miRNA
• 5 Housekeeping Genes
• Genomic DNA Contamination Control
• Reverse Transcription Controls (RTC) n=3
• Positive PCR Controls (PPC) n=3
Supported PCR Array Formats• 96-Well Plate• 384-Well Plate• 100-Well Disc• 96x96 Chip
miR-21 Targets PCR Array
Anatomy of a RT2 Profiler microRNA Targets PCR Arrays
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Sample & Assay Technologies- 35 -
Human Mouse RatmiR-21 miR-21 miR-21
miR-155 miR-155
miR-17 & miR-20a miR-17 & miR-20a miR-17 & miR-20a
miR-146a miR-146a miR-146a
miR-1 & miR-206 miR-1a & miR-206 miR-1 & miR-206
miR-34a miR-34a miR-34a
miR-221 & miR-222 miR-221 & miR-222 miR-221 & miR-222
let-7 let-7 let-7
Available RT² Profiler microRNA Targets PCR Arrays:
Custom miRNA targets arrays are possible based on your miRNA of interest
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Sample & Assay Technologies- 36 -
How RT2 Profiler PCR Arrays Work
. cDNA Synthesis (C-03 kit)– Genomic DNA Removal Step (5 min.)– Reverse Transcription Step (20 min.)
. Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet
. Run 40 cycle qPCR Program Standard cycling conditions for all
Real Time PCR Instruments 2 hours
. Upload and Analyze Data 15 minutes from Raw Ct to Fold Change Data
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Sample & Assay Technologies- 37 -
Data Analysis
. Web-Based Software No installation or licensing needed
. From Raw Ct Values to Fold Change Results ∆∆C(t) Method
. Multiple Analysis Formats Scatter Plot Volcano Plot Multi-Group Plot Clustergram
Also available as downloadable Excel templates
Scatter Plot
Volcano Plot
Clustergram
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Sample & Assay Technologies
Summary
Global miRNA function can by manipulated by using mimics and inhibitors
Specific miRNA/mRNA interactions can be studied using target protectors
Pathway or transcription factor assays can be used to follow miRNA affects onOn cell biology
RT2 miRNA Targets PCR Arrays facilitate screening miRNA target genes using verified qPCR Assays
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Sample & Assay Technologies- 39 -
Questions, Comments, Concern or Suggestions?
Answers to your technical and biological questions:
Technical Support: 888-503-3187 [email protected]
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 questions box.
miRNA mimics, inhibitors or target protectorsCignal reporter assayscDNA panelsmiRNA Targets RT2 Profiler PCR Arrays