Functional analysis of miRNA
Samuel J. Rulli, Jr. Ph.D.
samuel.rulli@QIAGEN.com

Sample & Assay Technologies
Topics for webinar
What is microRNA?
Manipulation of miRNA in cell lines using reporter
assays
miRNA Mimics
Inhibitors
Tar...
What Is Micro RNA?

RISC

Target mRNA

5’
CDS

3’

3’

5’
miRNA

Seed region

Endogenously expressed small functional RNAs...
Canonical pathway of microRNA biogenesis
Transcribed by RNA Polymerase II as
a long primary transcript (pri-miRNAs),
which...
A growing picture of miRNA dysregulation in cancer,
developmental and metabolic pathways
In the last 5 years, a substantia...
Topics for webinar
What is microRNA?
Manipulation of miRNA in cell lines using reporter
assays
miRNA Mimics
Inhibitors
Tar...
Manipulation of miRNA in cell lines
Functional interactions between miRNA and mRNA can be follow using:
Reporter assays: r...
miRNA Mimics & Inhibitors: How do they work?

Transfection of
Mimics

Transfection of
Inhibitors

RISC

Target
Expression
...
miRNA Mimics & Inhibitors: How do they work?

Transfection of
Mimics

Transfection of
Inhibitors

Mimics
increase
miRNA
ac...
Comparable down-regulation by mimics and endogenous miRNAs

Hela cells with reporter assays for miR-1 or miR-16. miR-16 is...
Stability of miRNA mimics

miRNA mimics are stable for at least 72 hours.
- 11 -

Sample & Assay Technologies
HDAC4 is a target of mir-1

HDAC4

HDAC4 had 2 miR-1 interaction sites. HeLa cells transfected with mir-1 show down
Regula...
miRNA Mimics & Inhibitors: How do they work?

Transfection of
Mimics

Transfection of
Inhibitors

Inhibitor decrease
miRNA...
mir-16 is decreased by miR-16 inhibitor in HeLa cells by reporter assay

- 14 -

Sample & Assay Technologies
Application Example: miRNA & Pathway Activity
The let-7 family of miRNAs is widely viewed as playing a role in tumor suppr...
Cignal Reporters: What You Need

• Cignal Cell Cycle Pathway Reporter
Measures E2F/DP1 Activity

• Transfection of HEK293 ...
Cignal Reporter Assays (45 Pathways)
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell...
Let-7 Family Members and the Cell Cycle

- 18 -

Sample & Assay Technologies
Topics for webinar
What is microRNA?
Manipulation of miRNA in cell lines using reporter
assays
miRNA Mimics
Inhibitors
Tar...
Approach to Identifying miRNA Regulators
miRNA mimic

miRNA

1

2

3

4

1) Treat Cells
2) Isolate RNA
3) First Strand cDN...
Transcriptome cDNA panels
Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the
miRNAs, that regulate ...
Example Transcriptome PCR Array

cDNA Sample 1
cDNA Sample 2
cDNA Sample 3
cDNA Sample 4
cDNA Sample 5
cDNA Sample 6

- 22...
Materials needed for miRNA discovery
.

.

.

Transcriptome PCR Array (1 for each GOI or HKG)
The Research Also Needs:
qPC...
SureFIND Protocol

- 24 -

Sample & Assay Technologies
miRNA pathways available

• Cancer miRNAs
• Immunopathology
• Development and differentiation
• Complete miRNome (~900 miR...
Application Example: miRNA regulation of INPPL1
INPPL1, also known as SHIP2, is a SH2-containing 5'-inositol phosphatase

...
Site Predictions for INPPL1

TargetScan

PicTar

miRANDA

miR-10

miR-184

miR-18a

miR-135

miR-205

miR-18b

miR-146

mi...
Cancer miRNA Transcriptome
PCR Array Results for INPPL1

- 28 -

Sample & Assay Technologies
Site Predictions for INPPL1

TargetScan

PicTar

miRANDA

miR-10

miR-184

miR-18a

miR-135

miR-205

miR-18b

miR-146

mi...
Compare Results with Research Community

What’s really exciting….
Three new miRNAs that play a role in INPPL1 expression
m...
Now what?............. Suggestions to Validate your “Hits”
miRNA-based Discovery
Perform a miScript miRNA mimic experiment...
Topics for webinar
What is microRNA?
Manipulation of miRNA in cell lines using reporter
assays
miRNA Mimics
Inhibitors
Tar...
Identification of transcriptional degradation using qPCR:
RT2 Profiler microRNA Targets PCR Arrays

Transfection of
Mimics...
Anatomy of a RT2 Profiler microRNA Targets PCR Arrays
miR-21 Targets PCR Array
•

84 Experimentally verified or
predicted ...
Available RT² Profiler microRNA Targets PCR Arrays:
Human
miR-21
miR-155

Mouse
miR-21
miR-155

Rat
miR-21

miR-17 & miR-2...
How RT2 Profiler PCR Arrays Work
cDNA Synthesis (C-03 kit)
– Genomic DNA Removal Step (5 min.)
– Reverse Transcription Ste...
Clustergram
Data

.

.

.

Analysis

Web-Based Software
Scatter Plot
No installation or licensing needed
From Raw Ct Value...
Summary

Global miRNA function can by manipulated by using mimics and inhibitors
Specific miRNA/mRNA interactions can be s...
Questions, Comments, Concern or Suggestions?

Answers to your technical and biological questions:
Technical Support:

888-...
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Mi rna functional analysis 2013

  1. 1. Functional analysis of miRNA Samuel J. Rulli, Jr. Ph.D. samuel.rulli@QIAGEN.com Sample & Assay Technologies
  2. 2. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session -2- Sample & Assay Technologies
  3. 3. What Is Micro RNA? RISC Target mRNA 5’ CDS 3’ 3’ 5’ miRNA Seed region 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 diseases e.g. Many different Cancers -3- Sample & Assay Technologies
  4. 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 -4- Sample & Assay Technologies
  5. 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 -5- Sample & Assay Technologies
  6. 6. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session -6- Sample & Assay Technologies
  7. 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 transcription Factors. -7- Sample & Assay Technologies
  8. 8. miRNA Mimics & Inhibitors: How do they work? Transfection of Mimics Transfection of Inhibitors RISC Target Expression Level -8- Sample & Assay Technologies
  9. 9. miRNA Mimics & Inhibitors: How do they work? Transfection of Mimics Transfection of Inhibitors Mimics increase miRNA activity RISC Target Expression Level -9- Sample & Assay Technologies
  10. 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 - 10 - Sample & Assay Technologies
  11. 11. Stability of miRNA mimics miRNA mimics are stable for at least 72 hours. - 11 - Sample & Assay Technologies
  12. 12. HDAC4 is a target of mir-1 HDAC4 HDAC4 had 2 miR-1 interaction sites. HeLa cells transfected with mir-1 show down Regulation of HDAC4 at the mRNA and protein levels. Sample & Assay Technologies
  13. 13. miRNA Mimics & Inhibitors: How do they work? Transfection of Mimics Transfection of Inhibitors Inhibitor decrease miRNA activity RISC Target Expression Level - 13 - Sample & Assay Technologies
  14. 14. mir-16 is decreased by miR-16 inhibitor in HeLa cells by reporter assay - 14 - Sample & Assay Technologies
  15. 15. 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 cycle progression and how? . - 15 - Sample & Assay Technologies
  16. 16. Cignal Reporters: What You Need • Cignal Cell Cycle Pathway Reporter Measures E2F/DP1 Activity • Transfection of HEK293 cells • Biological Test •Let-7 family mimics • Dual-Luciferase Assay - 16 - Sample & Assay Technologies
  17. 17. Cignal Reporter Assays (45 Pathways) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 GLI HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR - 17 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ Vitamin D Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR Sample & Assay Technologies
  18. 18. Let-7 Family Members and the Cell Cycle - 18 - Sample & Assay Technologies
  19. 19. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session - 19 - Sample & Assay Technologies
  20. 20. Approach to Identifying miRNA Regulators miRNA mimic miRNA 1 2 3 4 1) Treat Cells 2) Isolate RNA 3) First Strand cDNA Synthesis 4) qPCR for Gene Expression - 20 - Sample & Assay Technologies
  21. 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. . - 21 - Sample & Assay Technologies
  22. 22. Example Transcriptome PCR Array cDNA Sample 1 cDNA Sample 2 cDNA Sample 3 cDNA Sample 4 cDNA Sample 5 cDNA Sample 6 - 22 - Sample & Assay Technologies
  23. 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 - 23 - Sample & Assay Technologies
  24. 24. SureFIND Protocol - 24 - Sample & Assay Technologies
  25. 25. miRNA pathways available • Cancer miRNAs • Immunopathology • Development and differentiation • Complete miRNome (~900 miRNAs) Sample & Assay Technologies
  26. 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 . - 26 - Sample & Assay Technologies
  27. 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 - 27 - Sample & Assay Technologies
  28. 28. Cancer miRNA Transcriptome PCR Array Results for INPPL1 - 28 - Sample & Assay Technologies
  29. 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 - 29 - Sample & Assay Technologies
  30. 30. Compare Results with Research Community What’s really exciting…. Three new miRNAs that play a role in INPPL1 expression mir-193b mir-138 mir-373 - 30 - Sample & Assay Technologies
  31. 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 . - 31 - Sample & Assay Technologies
  32. 32. Topics for webinar What is microRNA? Manipulation of miRNA in cell lines using reporter assays miRNA Mimics Inhibitors Target protectors Screening your target gene for miRNA regulation Ready-to-use cDNA panels Identification of miRNA regulation by qPCR miRNA Targets PCR Arrays Summary Q & A session - 32 - Sample & Assay Technologies
  33. 33. Identification of transcriptional degradation using qPCR: RT2 Profiler microRNA Targets PCR Arrays Transfection of Mimics Transfection of Inhibitors RISC Target Expression Level - 33 - Sample & Assay Technologies
  34. 34. Anatomy of a RT2 Profiler microRNA Targets PCR Arrays miR-21 Targets PCR Array • 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 - 34 - Sample & Assay Technologies
  35. 35. Available RT² Profiler microRNA Targets PCR Arrays: Human miR-21 miR-155 Mouse miR-21 miR-155 Rat miR-21 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 Custom miRNA targets arrays are possible based on your miRNA of interest - 35 - Sample & Assay Technologies
  36. 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 . - 36 - Sample & Assay Technologies
  37. 37. Clustergram Data . . . Analysis Web-Based Software Scatter Plot 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 Volcano Plot Also available as downloadable Excel templates - 37 - Sample & Assay Technologies
  38. 38. 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 on On cell biology RT2 miRNA Targets PCR Arrays facilitate screening miRNA target genes using verified qPCR Assays Sample & Assay Technologies
  39. 39. Questions, Comments, Concern or Suggestions? Answers to your technical and biological questions: Technical Support: 888-503-3187 support@sabiosciences.com Samuel Rulli Samuel.Rulli@QIAGEN.com Copy of slides? Type slides into questions box or fill out survey on exit Want someone to call you? Type contact me into questions box Want to try something? Request demos in the questions box. miRNA mimics, inhibitors or target protectors Cignal reporter assays cDNA panels miRNA Targets RT2 Profiler PCR Arrays - 39 - Sample & Assay Technologies
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