Sample & Assay Technologies Meeting the challenges of miRNA research: microRNA Biogenesis, Function, and Analysis Jonathan Shaffer, Ph.D. [email protected]microRNA Technologies, R&D Americas The products described in this webinar are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of disease.
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The products described in this webinar are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of disease.
Sample & Assay Technologies Three part webinar series
Webinar 1: Meeting the Challenges of miRNA Research 2
miRNA and its role in human disease
Webinar 2 : Advanced microRNA expression analysis: From experimental design through data analysis
Speaker: Jonathan Shaffer, Ph.D.
Webinar 3 : Profiling miRNA expression in Cells, FFP E, and serum:On the road to biomarker development
Speaker: Jonathan Shaffer, Ph.D.
Webinar 1 : Meeting the challenges of miRNA research :An introduction to microRNA biogenesis, function, a nd analysis
Speaker: Jonathan Shaffer, Ph.D.
Sample & Assay Technologies Upcoming webinars
Webinar 1: Meeting the Challenges of miRNA Research 3
April 2013
Sign up at http://www.SABiosciences.com/seminarlist .php
Sample & Assay Technologies Meeting the Challenges of miRNA Research
Webinar 1: Meeting the Challenges of miRNA Research 4
Agenda
� miRNA Background
� miRNA Genomics
� miRNA in Disease
� miRNA Isolation Technologies
� miRNA Quantification Technologies
� miRNA Profiling Technologies
� miRNA Functionalization Technologies
Sample & Assay Technologies RNA interference: A natural phenomenon
Webinar 1: Meeting the Challenges of miRNA Research 5
Discovery tool, potential diagnostic, potential the rapeutic
� Changes in miRNA can be correlated with gene expression changes in development, differentiation, signal transduction, infection, aging, and disease.
Sample & Assay Technologies Canonical pathway of miRNA biogenesis
Webinar 1: Meeting the Challenges of miRNA Research 6
� 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.
Sample & Assay Technologies How do miRNAs interact with mRNAs?
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Basis of miRNA-mRNA interaction
Seed region: nucleotides 2-8 in 5’ region of miRNA� Most evolutionary conserved miRNA region� Most frequently complementary to target 3’-UTRs� Often sufficient to confer mRNA recognition
Beyond the seed region� 3’ end also contributes (extensive pairing is rare)� Some cases: central 11-12 continuous base pairs
Result of interaction� Suppression of gene expression� Rare cases: increase gene expression
References� Grimson, A., et al, Mol. Cell 2007, 27, 91-105 � Image From Bartel, D.P., Cell 2009, 136, 215-233� Guo, H., et al, Nature 2010, 466, 835-840� Thomson, D.W., et al, Nucleic Acids Res 2011, 1-9
Sample & Assay Technologies How to determine miRNA-mRNA interactions
Webinar 1: Meeting the Challenges of miRNA Research 8
Loss of miRNA Binding Site in targetSNP or MutationAlternative SplicingLoss/Change of 3’-UTR
Dicer Processing
Sample & Assay Technologies Unique signatures in human cancer
Webinar 1: Meeting the Challenges of miRNA Research 16
� miRNAs located in genomic regions amplified in cancers (e.g. miR-17-92 cluster) can function as oncogenes, whereas miRNAs located in portions of chromosomes deleted in cance rs (e.g. miR-15a-miR-16-1 cluster) can function as tumor suppressors.
� Abnormal expression of miRNAs has been found in both solid and hematopoietic tumors.
� miRNA expression fingerprints correlate with clinical and biological characteristics of tumors , including tissue type, differentiation, aggression and response to therapy.
In the last 10 years, a substantial number of studi es and reviews have associated the presence of various miRNAs with cell proliferation,
resistance to apoptosis, invasiveness, and differen tiation in cancer cells.
Sample & Assay Technologies miRNA in cancer diagnosis
Webinar 1: Meeting the Challenges of miRNA Research 17
Paranjape et al. (2009)
Gut. 58: 1546-1554.
Utilizing a signature of altered miRNA expression to
differentiate cancer tissue from normal tissue
Profiling circulating blood or tumor derived exosomalmiRNAs, surpassing the
invasive procedures to aid in early detection of cancers
Study SNPs in the miRNA genes, miRNA binding sites in the target
mRNA genes or in the miRNA processing/machinery pathway genes
to predict cancer predisposition
Use of miRNA based classifier to identify tissue
of origin for cancers of unknown primaries
Distinguishing tumor subtypes using a panel of
miRNAs that show differential expression within one cancer type
miRNAs in Cancer Diagnosis
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 18
Webinar 1: Meeting the Challenges of miRNA Research 19
Flexible protocols: total RNA or enrichment of miRN A
.High quality, pure RNA � Suitable for sensitive downstream
applications
.Rapid procedure � Streamlined protocol for low-
throughput or 96-well formats� Automated on QIAcube
Effective purification of total RNA� From a broad range of cells & tissues
.Efficient enrichment of microRNAs� miRNA enriched fraction & total RNA
separately� Co-purification of miRNA & total RNA
Sample & Assay Technologies miRNeasy FFPE Kit
Webinar 1: Meeting the Challenges of miRNA Research 20
High quality total RNA from archival samples
500 million FFPE tissues are archived!� Tissue banks, pathology labs, biomedical research labs
Current Isolation Methods: Compromised quality and yield� Heavily fragmented� Cross-linking by formaldehyde interferes with RT� Current procedures may not purify all usable RNA, may increase
fragmentation, are often ineffective in breaking up cross-links
.miRNeasy FFPE Kit: High quality, pure total RNA� Novel method prevents cross-linked RNA from blocking
downstream applications� Optimized lysis buffer with proteinase K� RNeasy MinElute for small elution volume
3. miScript PreAMP Kit� Optional step for small or precious samples� Full miRNome profiling from as little as 1 ng RNA
4. Assays� miScript Primer Assays� miScript Precursor Assays� QuantiTect Primer Assays
5. miScript SYBR Green PCR Kit� QuantiTect SYBR Green PCR Master Mix� Universal Primer
6. miScript miRNA PCR Array data analysis software � Straightforward, free data analysis
Sample & Assay Technologies miScript II RT Kit
Webinar 1: Meeting the Challenges of miRNA Research 24
A complete miRNA quantification solution
miScript II RT Kit
Biogenesis studies?Mature miRNA
quantification and profiling?
HiFlex Buffer HiSpec Buffer
Flexible detection of all RNA molecules
Patent-Pending technology for the specific detection of mature miRNAs
Note: Only HiSpec Buffer is recommended for use with miScript miRNA PCR Arrays
Sample & Assay Technologies HiFlex Buffer or HiSpec Buffer:
Webinar 1: Meeting the Challenges of miRNA Research 25
Which buffer should you choose?
HiFlex Buffer HiSpec Buffer
0
5
10
15
20
25
30
35
GA
PD
H
PPIA
CD
K1
MA
PK1
Let-
7a
miR
-16
miR
-21
miR
-25
Mean CT
0
5
10
15
20
25
30
35
GA
PD
H
PPIA
CD
K1
MA
PK
1
Let-
7a
miR
-16
miR
-21
miR
-25
Mean CT
Experiments that demand quantification of both mature miRNAs & mRNAs.
Experiments designed exclusively for mature miRNA quantification. Long RNAs, such as mRNAs, are not converted into cDNA. HiSpec is the optimized buffer for mature miRNA profiling with miScript miRNA PCR Arrays.
Sample & Assay Technologies miScript II RT Kit
Webinar 1: Meeting the Challenges of miRNA Research 26
Reverse transcription theory
Mix RNA, 5x miScript HiFlex or HiSpec Buffer, RNase-free water, 10x Nucleics Mix, and miScript
Reverse Transcriptase Mix Incubate at 37 °C for 60 min Incubate at 95 °C for 5 min
Use the cDNA to set up real-time PCR
reactions
miScript II Reverse Transcription Procedure
HiFlex Buffer HiSpec Buffer
Sample & Assay Technologies miScript II RT: Exceptional Linearity
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Linear over 6 logs of input RNA
10
15
20
25
30
35
40
-2 -1 0 1 2 3 4
Log (ng) of RNA in cDNA synthesis using the HiSpec Buffer
Mean CT
miR-16
miR-20a
miR-21
Linear (miR-16)
Linear (miR-20a)
Linear (miR-21)
10
15
20
25
30
35
40
-2 -1 0 1 2 3 4
Log (ng) of RNA in cDNA synthesis using the HiFlex Buffer
Mean CT
miR-16
miR-20a
miR-21
Linear (miR-16)
Linear (miR-20a)
Linear (miR-21)
HiFlex Buffer HiSpec Buffer
Sample & Assay Technologies miScript II PCR: Exceptional Sensitivity
Webinar 1: Meeting the Challenges of miRNA Research 28
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HCT 116 colorectal cancer cells
Scatter Plot Volcano Plot
1.E-05
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
1.E+01
1.E+02
1.E
-05
1.E
-04
1.E
-03
1.E
-02
1.E
-01
1.E
+00
1.E
+01
1.E
+02
2-∆CT
: Untreated
2-∆CT: Treated
� 5-aza-2’-dC irreversibly inhibits DNA methyltransferase driven DNA methylation reactions by incorporating into DNA and covalently binding to the active site of the DNMT.
� Scatter Plot: Significant differences exist in the mature miRNA expression levels of the two samples that were tested
� 104 miRNAs were strongly up-regulated in 5-aza-2’-dC treated cells, while 30 were strongly down-regulated in 5-aza-2’-dC treated HCT 116 cells.
� Volcano Plot: When a p Value of 0.05 is applied, the expression up-regulation of 89 of the 104 miRNAs is significant, and the expression down-regulation of 21 of the 30 miRNAs is significant
0.00001
0.0001
0.001
0.01
0.1
1
-8 -6 -4 -2 0 2 4 6 8
Log2 (Fold-Regulation)
p Value
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 45
Limiting Samples
Functionalization
Isolation
Quantification and Profiling
Body fluidsFFPE samplesLCM samplesFlow-sorted cellsCirculating tumor cellsFine needle biopsies
Sample & Assay Technologies miScript PreAMP: Excellent result, a fraction of th e input
Webinar 1: Meeting the Challenges of miRNA Research 47
Limiting FFPE sample
y = 0.9934x - 0.1457
R2 = 0.9606
-5
-3
-1
1
3
5
7
9
11
13
15
-5 -3 -1 1 3 5 7 9 11 13 15
∆CT: No PreAMP
∆CT: PreAMP
� Workflow� No PreAMP: 250 ng total RNA, HiSpec Buffer � Human miRNome miScript miRNA PCR Array (Plate 1)� PreAMP: 10 ng total RNA, HiSpec Buffer � one-tenth of cDNA, miScript PreAMP � preamplified cDNA
used for Human miRNome miScript miRNA PCR Array (Plate 1)
� Excellent correlation, PreAMP vs. No PreAMP� Preamplified cDNA provides enough material for more than 2 full human miScript
miRNome profiles. Input was only 1 ng of cDNA!
Sample & Assay Technologies miScript PreAMP: Excellent result, a fraction of th e input
Webinar 1: Meeting the Challenges of miRNA Research 48
Limiting serum sample
1.E-01
1.E+00
1.E+01
1.E+02
1.E+03
1.E
-01
1.E
+00
1.E
+01
1.E
+02
1.E
+03
2-∆CT
: Normal Serum
2-∆CT: Colorectal Cancer Serum
� Workflow� 5 µl serum � 14 µl total RNA � 2 µl total RNA, HiSpec Buffer � one-tenth of cDNA, miScript PreAMP �
preamplified cDNA (0.07 µl SE) used for Human Serum & Plasma 384HC miScript miRNA PCR Array
� Conclusion: Starting with only 5 µl of serum, significant differences were detected when colorectal cancer serum was compared to normal serum
y = 0.9728x + 0.7671
R2 = 0.9757
16
20
24
28
32
16 20 24 28 32
CT: Individual RNA Isolation from Serum
CT Mean: 3 RNA Isolations from Serum
Reproducibility 2 -∆CT: Colorectal Cancer vs. Normal
Sample & Assay Technologies Let miScript drive your miRNA studies!
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� 1 hour
� 2 minutes
� 2 hours
� 15 minutes
Sample
Results!
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 50
miRNA Functionalization Technologies
Functionalization
Isolation
Quantification and Profiling
Sample & Assay Technologies Manipulating miRNA function
Webinar 1: Meeting the Challenges of miRNA Research 51
Sample & Assay Technologies miScript miRNA Mimics
Webinar 1: Meeting the Challenges of miRNA Research 52
Mock endogenous miRNA activity
Features of miScript miRNA Mimics:� Double-stranded RNA oligonucleotide� Functional sequence is the same as the natural mature miRNA� Transfection results in inhibition comparable to the endogenous miRNA� Stable in culture for up to 72 hours
Webinar 1: Meeting the Challenges of miRNA Research 53
Block endogenous miRNA activity
Features of miScript miRNA Inhibitors:� Single-stranded, chemically modified RNA oligonucleotide� Designed and modified to ensure efficient inhibition of endogenous miRNA� Transfection of inhibitor counteracts miRNA-induced silencing � Effective for at least 96 hours following transfection
Sample & Assay Technologies miScript miRNA Mimics and Inhibitors
Webinar 1: Meeting the Challenges of miRNA Research 54
Controls
Mimic negative control
AllStars Negative Control siRNA� No homology to any mammalian gene.� Validated for non-effects in microarray and phenotype assays.� Validated for RISC entry
Mimic positive control
Syn-hsa-miR-1 mimic� Not expressed under most culture conditions, only expressed in muscle cells � To check for optimal conditions by using miScript PCR miR-1
Inhibitor negative control
miScript Inhibitor Negative Control� Target the sequence of miScript mimic negative control
Inhibitor positive control
Anti-hsa-miR-1 inhibitor � To check for optimal conditions in combination with Syn-hsa-miR-1 mimic
Transfection control
AllStars Hs Cell Death Control siRNA� Cell death = successful transfection� To run in every experiment
Webinar 1: Meeting the Challenges of miRNA Research 55
What genes are regulated by your miRNA of interest?
Benefits:� Assist miRNA functional analysis� Follow up on miRNA expression profiling analyses� Species currently covered: Human, mouse, and rat
Features:� Profiles expression of 84 verified & bioinformatically predicted target genes� Target genes specific for a miRNA & others in same seed sequence family� Free data analysis suite� Available in 2, 12 or 24 plate packs� Compatible with any real time qPCR instrument currently in your lab
Note: Uses RT 2 qPCR Reagent Kits
Sample & Assay Technologies Where can I find miScript miRNA PCR Arrays?
Webinar 1: Meeting the Challenges of miRNA Research 56
www.sabiosciences.com/mirna_pcr_array.php
� miRNA Overview
� miScript PCR System
� miScript miRNA PCR Arrays
� Products for functional studies
� miRNA purification options
Sample & Assay Technologies Where can I find miScript Primer Assays?
Webinar 1: Meeting the Challenges of miRNA Research 57