1. Functional analysis of miRNA
Samuel J. Rulli, Jr. Ph.D.
samuel.rulli@QIAGEN.com
Sample & Assay Technologies
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
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Sample & Assay Technologies
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
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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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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
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
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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 transcription
Factors.
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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
Mimics
increase
miRNA
activity
RISC
Target
Expression
Level
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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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11. Stability of miRNA mimics
miRNA mimics are stable for at least 72 hours.
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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. miRNA Mimics & Inhibitors: How do they work?
Transfection of
Mimics
Transfection of
Inhibitors
Inhibitor decrease
miRNA activity
RISC
Target
Expression
Level
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14. mir-16 is decreased by miR-16 inhibitor in HeLa cells by reporter assay
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Sample & Assay Technologies
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?
.
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Sample & Assay Technologies
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
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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
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
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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
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
25. miRNA pathways available
• Cancer miRNAs
• Immunopathology
• Development and differentiation
• Complete miRNome (~900 miRNAs)
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
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 expression
mir-193b
mir-138
mir-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
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
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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
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
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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. 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
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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. 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
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Sample & Assay Technologies
