1. Sample & Assay Technologies
miRNA Profiling:
On the Road to Biomarker Development
Jonathan Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.com
Scientist, miRNA Technologies
Research & Development
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.
2. Sample & Assay Technologies
Three part webinar series
miRNA and its role in human disease
Webinar 1 :
Speaker:
Webinar 2 :
Speaker:
Webinar 3 :
Speaker:
Meeting the challenges of miRNA research:
An introduction to microRNA biogenesis, function, and analysis
Jonathan Shaffer, Ph.D.
Advanced microRNA expression analysis:
From experimental design through data analysis
Jonathan Shaffer, Ph.D.
Profiling miRNA expression in Cells, FFPE, and serum:
On the road to biomarker development
Jonathan Shaffer, Ph.D.
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
2
3. Sample & Assay Technologies
Profiling miRNA expression in cells, FFPE, and serum
Agenda
Introduction to miRNA and disease
Where and how do you access the information?
Samples and sample prep options
Profiling miRNA expression using the miScript PCR System
System components, options, and workflows
Summary of the miScript PCR System
Questions
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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4. microRNA (miRNA) timeline
Sample & Assay Technologies
Natural phenomenon, discovery tool, potential biomarker
dsRNA, Dicer
Flys -Zamore
miRNA in cancer
Croce lab
Antagomirs
Stoffel lab
miRNA as serum
biomarker, Tewari
PTGS in plants
Baulcombe lab
Discovery of
Lin-4, Ambros
Large numbers of miRNA
Bartel, Tuschl, Ambros
Discovery of
Let-7, Ruvkin
RNAi in C. elegans
Fire and Mello
1993
1997
1998
1999
miRNA as Dx, ProgDx
Takamizawa
50% miRNA in
cancer regions, Calin
RNAi in mammalian
cells, Tuschl lab
2000
2001
2002
2003
miRNA and
epigenetics
Saito
miRNA as cancer
therapy, Kota et al
miRNA molecular
decoys
Eiring et al.
Transcriptional
activation, Li et al.
2004
2005
2006
2007
2008
2009
Direct miRNA-target
in cancer, Slack lab
miRNA as oncogene
in vivo, Hammond lab
Science Magazine:
Breakthrough
of the Year
2010
Cancer by single
miRNA, Slack lab
miRNA and SNPs
Shen et al.
FORTUNE Magazine:
Biotech’s Billion Dollar
Breakthrough
Nobel Prize: Fire
and Mello
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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5. Sample & Assay Technologies
Why quantify miRNA?
Virtually every publication includes characterization by quantification.
Changes in miRNA can be correlated with gene expression changes in development,
differentiation, signal transduction, infection, aging, and disease.
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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6. Sample & Assay Technologies
Canonical pathway of miRNA 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.
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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7. Sample & Assay Technologies
Potential events that disrupt normal miRNA activity
Disruption of miRNA–mRNA interaction
Altered transcription
Genomic instability
Methylation
Histone modification
Transcription factor
Amplification/deletion
Translocation
Insertional mutagenesis
Drosha processing
Dicer processing
Loss of miRNA
binding site in target
SNP or mutation
Alternative splicing
Loss/change of 3′-UTR
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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8. Sample & Assay Technologies
miRNA Isolation Technologies
Isolation
Quantification
and Profiling
Functionalization
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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9. Sample & Assay Technologies
miRNeasy Kits: Mini, Micro
Flexible protocols: total RNA or enrichment of miRNA
High quality, pure RNA
.
Suitable for sensitive downstream
applications
Rapid procedure
.
Streamlined protocol for lowthroughput 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
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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10. Sample & Assay Technologies
miRNeasy FFPE Kit
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
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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11. Sample & Assay Technologies
miRNeasy Serum/Plasma Kit
Purification from serum, plasma, and other fluids
For purification of circulating RNA from
animal and human plasma and serum
Includes synthetic RNA control assay for normalization
Clarified plasma
or serum
Minimal elution volume (14 µl)
High-purity RNA suitable for all downstream applications
miScript RT: up to 10 µl eluate for cDNA synthesis
One RT enough for 6, 384-well plates
Easy, robust procedures
Plasma
QIAzol
Bind
Serum
Wash
Elute
Manual or Automated on QIACube
Automatable protocol
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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12. Sample & Assay Technologies
miRNA in Blood
RBC, WBC, platelets, CTC, ‘other cells’, extracellular?
What is blood?
RBC, WBC,
platelets, other cells
(e.g. circulating tumor cells)
Serum (post clotting)
Plasma (no-clotting)
High levels of nucleases present in plasma
Freely circulating RNA should be rapidly degraded
Surprisingly, stabile miRNA can be detected in serum and plasma
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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13. Sample & Assay Technologies
Stable miRNA in circulation ~2007
An evolving story
Ago
miRNA
Exosomes &
micro vesicles1,2,3
Ago-2-miRNA
complexes4
HDL mediated
miRNA transport5
Other ‘protective’
protein6
1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange
between cells, Nat Cell Biol 9:654-659
2) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e3694
3) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 1744217452
4) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human
plasma, Proc. Natl. Acad. Sci 108: 5003-5008
5) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.
Nat Cell Biol 13:423
6) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian
cells. Nucleic Acids Res. 2010 Nov 1;38(20):7248-59.
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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15. Sample & Assay Technologies
miRNA in cancer diagnosis
Use of miRNA based
classifier to identify tissue
of origin for cancers of
unknown primaries
Utilizing a signature of altered
miRNA expression to
differentiate cancer tissue
from normal tissue
Profiling circulating blood or
tumor derived exosomal
miRNAs, surpassing the
invasive procedures to aid in
early detection of cancers
miRNAs
in Cancer
Diagnosis
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
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
Distinguishing tumor
subtypes using a panel of
miRNAs that show
differential expression
within one cancer type
Paranjape et al. (2009)
Gut. 58: 1546-1554.
15
16. Sample & Assay Technologies
miRNA Quantification Technologies
Isolation
Quantification
and Profiling
Functionalization
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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17. Sample & Assay Technologies
miScript PCR System
Complete miRNA quantification system
1.
miScript II RT Kit
HiFlex Buffer: Unparalleled flexibility for quantify miRNA
and mRNA quantification from a single cDNA preparation
HiSpec Buffer: Unmatched specificity for mature
miRNA profiling
2.
miScript miRNA PCR Arrays
miRNome
Pathway-focused
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 MM
Universal Primer
6.
miScript miRNA PCR Array data analysis software
Straightforward, free data analysis
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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18. Sample & Assay Technologies
miScript II RT Kit
One box with 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
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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19. miScript II RT Kit
Reverse transcription using HiSpec Buffer
Linearity: 2 µg to 10 pg
40
miR-16
miR-20a
35
miR-21
Mean CT
30
25
20
15
10
-2
-1
0
1
2
3
Log (ng) of RNA in cDNA synthesis using HiSpec Buffer
4
Sensitivity: 1x106 copies to 10 copies
32
miR-21
28
Mean CT
Sample & Assay Technologies
24
20
16
12
1
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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3
4
5
Log copy number of miRNA using HiSpec Buffer
6
19
20. Sample & Assay Technologies
miRNA Profiling Technologies
Isolation
Quantification
and Profiling
Functionalization
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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21. Sample & Assay Technologies
miRNA expression profiling applications
Mechanisms of gene regulation
Developmental biology
Novel miRNA discovery
Studying miRNA–mRNA and miRNA–protein interactions
Integrative analyses of miRNAs in the context of gene
regulatory networks
Biomarkers
Tissue-based miRNA biomarkers
Tissues of unknown origin
Circulating biomarkers
Forensics
From Pritchard, C.C., et al, Nature Rev. Genet 2012, 13, 358-369
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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22. Sample & Assay Technologies
miScript miRNA PCR Arrays
Pre-formatted solutions for miRNA profiling experiments
Pre-formatted, single use PCR arrays with wet-lab verified assays
Fully customizable (assays and species)
miRNome Arrays
Most species
Largest content
High Content (HC) Arrays
Targeted miRNome profiling
Focused Arrays
Biological relevant
Intelligently designed
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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23. Sample & Assay Technologies
miRNome miScript miRNA PCR Arrays
Leading coverage and validated assays
miRNome Arrays
Human
Mouse
Rat
Dog
Rhesus macaque
Species
Assays
(miRBase V16)
Human
940
Rat
653
Dog
277
Rhesus macaque
469 (V18)
100% validated assays
Each assay is bench validated
Each array is quality controlled
Leading miRNome coverage
Customizable
1066
Mouse
Benefits of miRNome Arrays
miRBase V17 and V18 assays are
available!
Contact us if you are interest in a
different species!
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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24. Sample & Assay Technologies
Focused miScript miRNA PCR Arrays
Biologically relevant, intelligently designed
Focused Arrays
miFinder
Cancer PathwayFinder
Brain Cancer
Breast Cancer
Ovarian Cancer
Liver miFinder – New!
Apoptosis
Cell Differentiation & Development
Immunopathology
Inflammatory Response & Autoimmunity
Diabetes
Neurological Development & Disease
T-Cell & B-Cell Activation
Prostate Cancer
Cardiovascular Disease
Serum & Plasma
Benefits of Focused Arrays
100% validated assays
Each assay is bench validated
Each array is quality controlled
Biological relevant and up-to-date
Customizable
Contact us if you are interest in a
different species!
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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25. Sample & Assay Technologies
High Content (HC) miScript miRNA PCR Arrays
Targeted miRNome profiling
miFinder 384HC
Serum & Plasma 384HC
Cancer PathwayFinder 384HC
Liver miFinder 384HC – New!
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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26. Sample & Assay Technologies
Anatomy of a miScript miRNA PCR Array
96-well Format: 84 miRNA + 12 controls
84 miRNAs
Cel-miR-39
SNORD61; SNORD68; SNORD72
SNORD95; SNORD96A; RNU6-2
miRTC
PPC
Spike in
Control
miScript PCR Controls for
Normalization
RT
Control
PCR
Control
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
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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27. Sample & Assay Technologies
miScript PCR Controls
Stable expression and excellent amplification efficiencies
Tissue Expression
35
Mean CT
30
SNORD61
SNORD68
25
SNORD72
20
SNORD95
15
SNORD96A
RNU6-2
10
5
Brain
Kidney
Liver
Lung
Skeletal
Muscle
Testis
Thymus
Amplification Efficiencies
Primer
Hsa
Mmu
Rno
Cfa
Mml
SNORD61
103
99
95
99
101
SNORD68
101
100
93
97
99
SNORD72
94
100
93
91
97
SNORD95
102
103
98
100
106
SNORD96A
102
95
95
92
104
RNU6-2
98
98
95
99
103
Highly conserved across multiple species
Human, Mouse, Rat, Dog, Rhesus macaque
Relatively stable expression in many tissues
Amplification efficiencies of these assays are 100%
Consistent performance in both HiSpec and HiFlex Buffers
Ideal normalizers for ∆∆CT method of relative quantification
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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28. Sample & Assay Technologies
miScript miRNA PCR Arrays
Formats built to fit your cycler AND your experiment
96-well
384-well
384-well (4 x 96)
Rotor-Disc 100
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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29. Sample & Assay Technologies
miScript miRNA PCR Arrays
Compatible 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
iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384
Opticon, Opticon 2, Chromo 4
Mastercycler ep realplex 2/2S/4/4S
LightCycler 480
Mx3000p, Mx3005p, Mx4000p
TP-800
RotorGene Q
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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30. Sample & Assay Technologies
miScript miRNA PCR Arrays
QIAGEN PCR Array Service Core
Send your samples and receive results!
Total RNA Isolation: miRNeasy Kits
Reverse Transcription: miScript II RT Kit
qPCR: miScript miRNA PCR Arrays
Data analysis included!
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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31. Sample & Assay Technologies
miScript miRNA PCR Arrays
Rapid Workflow = Robust, Reproducible Performance
1st Time Array User
1 hour
HiSpec Buffer
2 minutes
2 hours
15 minutes
Total HeLa S3 (miRNeasy)
Pellet 1: Frozen June 2010
Pellet 2: Frozen April 2011
HiSpec Buffered cDNA
miScript real-time PCR
miFinder miScript miRNA PCR Array
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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32. Sample & Assay Technologies
miScript PreAMP PCR Kit: decrease input up to 1000X
An entire miRNome from only 10 ng of total RNA
Operator Variability Assessment
Universal reference RNA
Two operators
Experienced operator
First time operator
miScript real-time PCR
miFinder miScript miRNA PCR Array
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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33. Sample & Assay Technologies
miRNome miRNA expression profiling
HCT 116 colorectal cancer cells
Scatter Plot
Volcano Plot
1.E+02
0.00001
0.0001
1.E+00
1.E-01
p Value
2
-∆CT
: Treated
1.E+01
1.E-02
1.E-03
0.001
0.01
1.E-04
0.1
1.E-05
1.E+02
1.E+01
1.E+00
1.E-01
1.E-02
1.E-03
1.E-04
1.E-05
2
1
-8
-6
-∆CT
: Untreated
-4
-2
0
2
4
6
8
Log2 (Fold-Regulation)
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 downregulated 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
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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34. Sample & Assay Technologies
Focused miRNA expression profiling
FFPE samples
Reproducibility
2-∆CT: Lung Tumor vs. Normal
One 5 µm FFPE section used per FFPE isolation
On average, each isolation provided enough total RNA for:
– Two full human miRNome profiles
– Ten pathway-focused PCR arrays
RT: 125 ng total RNA, HiSpec Buffer
qPCR: Human miFinder miScript miRNA PCR Array (0.5 ng cDNA per well)
Significant differences exist between the miRNA expression levels of the two tissue types
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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35. Sample & Assay Technologies
miScript PreAMP: Excellent result with 250-fold less input
Limiting FFPE sample
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!
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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36. Sample & Assay Technologies
-80° vs. RT for 24 hrs
C
miRNA Expression Profiling from Body Fluids
Freeze-thaw cycles
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
Plasma vs. Serum
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37. Sample & Assay Technologies
Workflow: Serum & plasma miRNA expression profiling
1. Collect whole blood and separate serum or plasma
2. Recommended starting amount of serum or plasma: 100–200 µl
Note: If starting with 50 µl (or less) of serum or plasma, incorporate preamplification
3. Isolate RNA: miRNeasy Serum/Plasma Kit + miRNeasy Serum/Plasma Spike-in Control
4. Reverse transcription: Perform miScript II RT reaction (using HiSpec Buffer)
Per intended 384-well plate, reverse transcribe 1.5 µl RNA eluate (1/10th prep)
5. (Optional) Preamplification: If the starting amount of serum or plasma is 50 µl (or less),
perform preamplification using the miScript PreAMP PCR Kit
6. Real-time PCR: Profile miRNA expression using chosen miScript miRNA PCR Array
Array recommendations:
– miRNome miScript miRNA PCR Array
– Serum & Plasma 384HC miScript miRNA PCR Array
– Serum & Plasma miScript miRNA PCR Array
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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38. Sample & Assay Technologies
miRNA expression profiling using serum samples
High Content (HC) Array: 384 assays
2-∆CT: Cancer vs. Normal
Reproducibility
Workflow
200 µl serum
14 µl total RNA
miRNA PCR Array
1.5 µl total RNA, HiSpec Buffer
Serum & Plasma 384HC miScript
Significant differences exist between the miRNA expression levels of the two serum types
± 3-fold [red lines] used as a cutoff for significance
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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39. Sample & Assay Technologies
miScript PreAMP: Excellent result with 300-fold less input
Limiting serum sample
Reproducibility
2-∆CT: Colorectal Cancer vs. Normal
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
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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40. Sample & Assay Technologies
Workflow: Urine miRNA expression profiling
Preamplification is highly recommended
No PreAMP: High CT values
PreAMP: Robust CT values
1. Recommended starting amount of urine: 200 µl
2. Isolate RNA: miRNeasy Serum/Plasma Kit + miRNeasy Serum/Plasma Spike-in Control
3. Reverse transcription: Reverse transcribe 1.5 µl RNA eluate (1/10th prep)
using a HiSpec buffered miScript II RT Kit reaction
4. Preamplification: Preamplify cDNA using the miScript PreAMP PCR Kit and the
miScript PreAMP Primer Mix of interest
5. Real-time PCR: Using amplified cDNA, profile miRNA using chosen miScript miRNA PCR Array
Array recommendations:
– miRNome miScript miRNA PCR Array
– miFinder 384HC miScript miRNA PCR Array
– miFinder miScript miRNA PCR Array
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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41. Sample & Assay Technologies
Urine miRNA profiling: PreAMP is recommended
Results are highly reproducible
Workflow
200 µl urine
14 µl total RNA 1.5 µl total RNA, HiSpec Buffer
one-tenth of cDNA, miScript
PreAMP
preamplified cDNA used for Human miFinder miScript miRNA PCR Array
Conclusion: Starting with only 200 µl urine, preamplification enables accurate, reproducible
miRNA expression profiling
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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42. Sample & Assay Technologies
Biomarker discovery workflow
Concept
Phase I (determined expressed miRNAs):
Pooled sample profiling,
Maximal Assays (miRNome or 384HC)
Phase II (determine differentially expressed miRNAs):
Individual profiling of samples (that went into pools),
Only Expressed miRNAs (< 384-well plate)
Phase III (statistical power):
Individual profiling of all samples in study,
Differentially Expressed miRNAs from Phase II (multiple samples per 384-well plate)
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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43. Sample & Assay Technologies
Biofluids: miRNA data normalization
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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44. Sample & Assay Technologies
Data normalization
Step 1
Check reverse transcription (miRTC) and PCR (PPC) controls
Position
Control
CT: Serum Sample A
CT: Serum Sample B
H09
miRTC
18.8
18.5
H10
miRTC
18.7
18.6
H11
PPC
19.4
19.6
H12
PPC
19.6
19.8
As determined by the raw CT values, the reverse transcription
and PCR efficiency of both samples are highly comparable
CT values differ by less than 0.25 units
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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45. Sample & Assay Technologies
Data normalization
Step 2
Check snoRNAs/snRNA controls
Control
CT: Serum Sample A
CT: Serum Sample B
SNORD61
36.3
34.3
SNORD68
34.6
35.0
SNORD72
35.0
35.0
SNORD95
31.1
39.3
SNORD96A
33.6
34.5
RNU6-2
37.9
39.1
snoRNAs/snRNAs exhibit borderline detectable expression
As is often found with serum samples, standard housekeeping
assays cannot be used for data normalization
In fact, this data indicated a prep free of cellular contamination
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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46. miScript PCR Controls
Sample & Assay Technologies
Strong expression in 100% blood, low expression in serum
100% Blood:
CT Mean
100% Serum:
CT Mean
∆CT
(Serum - Blood)
RNU6-2
21.9
34.4
12.5
SNORD61
24.6
35.4
10.8
SNORD68
20.7
33.4
12.7
SNORD96A
23.1
34.1
11.0
SNORD95
21.6
30.9
9.3
SNORD72
27.0
35.1
8.1
Target
36
Blood Percentage
(%)
Mean CT
cel-miR-39
19.24
23.52
50
28
CT Mean:
miScript Controls
100
32
CT Mean:
cel-miR-39
18.92
24.41
10
18.08
26.37
18.70
30.03
19.91
33.52
0.01
19.42
33.75
0.001
19.67
33.94
0.0001
20
1
0.1
miScript Controls
24
19.03
33.74
16
100
50
10
1
0.1
0.01
0.001 0.0001
Blood Percentage (%)
If the mean CT of the miScript PCR Control panel is lower than 32, then
you have greater than 0.1% cellular contamination from cells
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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47. Sample & Assay Technologies
Data normalization
Step 3
Calibrate data of each plate to its RNA Recovery Controls
Can only be used if Syn-cel-miR-39 miScript miRNA Mimic is spiked into the
sample during RNA preparation
Then: If your array is ‘unbiased’ (full miScript PCR Array)
Normalize to the CT mean of all expressed targets on plate (OK)
OR
Normalize to the CT mean of commonly expressed targets on plate
(better)
Or: If your array is ‘biased’ (small panel of regulated miRNAs)
Normalize data to CT of several invariant miRNAs
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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48. Sample & Assay Technologies
Where can I find miScript miRNA PCR Arrays?
www.sabiosciences.com/mirna_pcr_array.php
miRNA Overview
miScript PCR System
miScript miRNA PCR Arrays
Products for functional studies
miRNA purification options
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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49. Sample & Assay Technologies
Where can I find miScript Primer Assays?
www.qiagen.com/GeneGlobe
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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50. Sample & Assay Technologies
RT2 Profiler miRNA Targets PCR Array
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 RT2 qPCR Reagent Kits
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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51. Sample & Assay Technologies
QIAGEN’s miRNA portfolio
Your miRNA workflow, from sample to results!
Quantification
and profiling
miRNeasy Mini Kit, miRNeasy Micro Kit
miRNeasy 96 Kit
miRNeasy FFPE Kit
miRNeasy Serum/Plasma Kit
Profiling
Isolation
Functionalization
miScript II RT Kit and PreAMP Kit
HiPerFect Transfection Reagent
miScript SYBR Green PCR Kit
Attractene Transfection Reagent
miScript miRNA PCR Arrays
miScript miRNA Mimics
miScript miRNA Data Analysis Tool
miScript miRNA Inhibitors
PAXgene Tissue miRNA Kit
miScript Primer Assay
Custom miScript miRNA Mimics
PAXgene Blood miRNA Kit
miScript Precursor Assay
miScript Target Protector
Supplementary protocol for miRNA from
Plasma and Serum
miScript PCR Starter Kit
miScript miRNA Inhibitor 96 and 384
Plates and Sets
QIAGEN Service Core
QIAcube
QIAgility
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
Rotor-Gene Q
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52. Sample & Assay Technologies
Upcoming webinars
May 2013
Sign up at http://www.SABiosciences.com/seminarlist.php
Webinar 3: Profiling miRNA expression in cells, FFPE, and serum
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53. Sample & Assay Technologies
Thank you for attending
Technical Support Contact Information
Monday through Friday
8:00AM to 6:00PM EST
Direct Phone: 888-503-3187
E-Mail: support@sabiosciences.com
http://www.sabiosciences.com/promotion/miscriptdemo.php
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54. Sample & Assay Technologies
Questions?
Thank you for attending today’s webinar!
Jonathan Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.com
microRNA Technologies, R&D Americas
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