Transcript
Sample & Assay Technologies
Meeting the challenges of miRNA research :
microRNA Biogenesis, Function, and Analysis
Jonathan Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.commicroRNA 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.
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
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� 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
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� 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
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Algorithms for predicting miRNA-mRNA interaction
Prediction Algorithm Website
TargetScan http://www.targetscan.org/
Pictar http://pictar.mdc-berlin.de/
MicroCosm Targets http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/
DIANA http://diana.cslab.ece.ntua.gr/microT/
miRANDA http://www.microrna.org/microrna/home.do
TarBase (experimentally supported)
http://diana.cslab.ece.ntua.gr/tarbase/
Target Prediction is based on:� Bioinformatics
� Seed region match� Position in 3’ UTR� Cross species conservation� Central sequence homology
� Wet lab research� Empirical evidence from microarrays� Reporter systems
Pitfalls of using prediction algorithms:� Large number of candidate mRNAs for a given
miRNA� May not incorporate all miRNA targeting
possibilities� Different algorithms produce different target
lists� Potential for false positive rate of prediction
Sample & Assay Technologies How to determine miRNA-mRNA interactions
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Experimental techniques
miRNA Target Screening� Gene expression analysis (inferred targets)
� RNA-seq� Microarrays� qPCR
� Immunoprecipitation (direct targets)� HITS-CLIP� PAR-CLIP� Biotin tagged miRNA
Gene-Specific Validation� qPCR� Luciferase reporter assays� Western blot� 5’ rapid identification of cDNA ends (5’ RLM-RACE)
Other techniques� Parallel analysis of RNA ends (modified 5’ RLM-RACE)� Reverse transcription of targets
Image From Chi, S.W., et al, Nature 2009, 13, 479-486
Sample & Assay Technologies
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miRNA Genomics: Generating miRNome Diversity
Functionalization
Isolation
Quantification and Profiling
Sample & Assay Technologies miRNA genomic structure
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� Intergenic miRNA genes : either monocistronic or polycistronic with a common promoter
� Intronic miRNA genes : present in the introns of protein coding or noncoding genes, can also be in clusters, transcribed by the host gene promoter
� Exonic miRNAs genes : rare and often overlap an exon and an intron of a noncoding gene
� miRNAs can be transcribed from the negative strand within or near a protein coding gene
Sample & Assay Technologies Multiple loci can generate the same mature miRNA
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Biogenesis creates incredible diversity
www.mirbase.org
Stem Loop CHR Overlapping transcripts CHR: Coordinat es (GRCh37)
1302-1 12 intergenic 12: 113132839-113132981 [-]
1302-3 2 intergenic 2: 114340536-114340673 [-]
1302-7 8 intergenic 8: 142867603-142867674 [-]
1302-10 15 intergenic 15: 102500662-102500799 [-]
1302-11 19 intergenic 19: 71973-72110 [+]
1302-2 1 intronic Non protein coding 1: 30366-30503 [+] sense
1302-4 2 intronic Non protein coding 2: 208133999-208134148 [-]
1302-9 9 Non protein coding 9: 30144-30281 [+]; Sense
1302-5 20 intronic Protein coding/FAM65C; intron 4 20: 49231173-49231322 [-]; Sense
1302-6 7 intronic Protein coding/HDAC9; intron 1 7: 18166843-18166932 [-] ; Antisense
1302-8 9 intronic Protein coding/ch9orf174 9: 100125836-100125963 [-]; Antisense
Mature-miR-1302: UUGGGACAUACUUAUGCUAAA
Sample & Assay Technologies Post-transcriptional modification of miRNA
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Processing and ADAR editingD
eep
sequ
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f hsa
-miR
-21-
5p
Pre-miRNA
Exportin
Pri-miRNA
ADAR
A�I
A����I DROSHA Processing Block
A����IDICER
Processing Block
Change in miRNA seedA����I
ADAR
ADAR
ADAR: adenosine deaminase, RNA specificA-> I destabilizes dsRNA
Sample & Assay Technologies
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miRNA in Disease
Functionalization
Quantification and Profiling
Isolation
Sample & Assay Technologies Potential events that disrupt normal miRNA activity
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Disruption of miRNA-mRNA interaction
Webinar 1: Meeting the Challenges of miRNA Research
Altered TranscriptionMethylation
Histone ModificationTranscription Factor
Drosha Processing
Genomic InstabilityAmplification/DeletionTranslocationInsertional Mutagenesis
Loss of miRNA Binding Site in targetSNP or MutationAlternative SplicingLoss/Change of 3’-UTR
Dicer Processing
Sample & Assay Technologies Unique signatures in human cancer
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� 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
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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
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miRNA Isolation Technologies
Functionalization
Isolation
Quantification and Profiling
Sample & Assay Technologies miRNeasy Kits: Mini, Micro
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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
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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
Sample & Assay Technologies miRNeasy Serum/Plasma Kit
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Purification from serum, plasma, and other fluids
� Includes synthetic RNA control assay for normalization
� 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
� Automatable protocol
QIAzol Bind Wash Elute
Manual or Automated on QIACube
Plasma Serum
Clarified plasmaor serum
For purification of circulating RNA from animal and human plasma and serum
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 22
miRNA Quantification Technologies
Functionalization
Isolation
Quantification and Profiling
Sample & Assay Technologies miScript PCR System
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Fully integrated, complete miRNA quantification sys tem
1. miScript II RT Kit� HiFlex Buffer: Unparalleled flexibility for 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 Master Mix� Universal Primer
6. miScript miRNA PCR Array data analysis software � Straightforward, free data analysis
Sample & Assay Technologies miScript II RT Kit
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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:
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Which buffer should you choose?
HiFlex Buffer HiSpec Buffer
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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
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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
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Log (ng) of RNA in cDNA synthesis using the HiSpec Buffer
Mean CT
miR-16
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Linear (miR-20a)
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Linear (miR-20a)
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HiFlex Buffer HiSpec Buffer
Sample & Assay Technologies miScript II PCR: Exceptional Sensitivity
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Detection of 10 copies to >10 6 copies of miRNA
HiFlex Buffer HiSpec Buffer
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Log copy number of miRNA using the HiSpec Buffer
Mean CT
miR-21
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Mean CT
miR-21
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Sample & Assay Technologies miScript PCR System: Exceptional Specificity
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Excellent discrimination between closely related miRNA family members
Relative detection (as % of perfect match)
cDNA used in PCR
miScript Primer Assay Used
Let-7b Let-7c miR-98 Let-7d Let-7e Let-7a Let-7f Let-7g Let-7i
Let-7b 100.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Let-7c 0.5 100.0 0.0 0.0 1.0 0.1 0.0 0.0 0.0
miR-98 0.0 0.2 100.0 0.1 0.0 0.1 0.0 0.0 0.1
Let-7d 0.1 0.0 0.0 100.0 0.0 0.4 0.0 0.0 0.0
Let-7e 0.1 0.0 0.0 0.0 100.0 0.2 0.0 0.0 0.0
Let-7a 0.1 0.6 0.0 0.5 3.9 100.0 0.1 0.0 0.0
Let-7f 0.6 0.1 0.0 0.1 0.0 1.1 100.0 0.1 0.1
Let-7g 0.6 0.2 0.0 0.1 0.0 0.0 0.0 100.0 0.2
Let-7i 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 100.0
HiFlex Buffer
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 30
miRNA Profiling Technologies
Functionalization
Isolation
Quantification and Profiling
Sample & Assay Technologies miRNA expression profiling applications
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� 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
Sample & Assay Technologies miRNome miScript miRNA PCR Arrays
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Leading coverage and validated assays
� Human� Mouse� Rat � Dog� Rhesus macaque
� 100% validated assays� Each assay is bench validated� Each array is quality controlled
� Leading miRNome coverage
� Customizable
� miRBase V17 and V18 assays are available!
� Contact us if you are interest in a different species!
miRNome Arrays Benefits of miRNome Arrays
Species Assays(miRBase V16)
Human 1066
Mouse 940
Rat 653
Dog 277
Rhesus macaque 469 (V18)
Sample & Assay Technologies Focused miScript miRNA PCR Arrays
Webinar 1: Meeting the Challenges of miRNA Research 33
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
� 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!
Benefits of Focused Arrays
Sample & Assay Technologies High Content (HC) miScript miRNA PCR Arrays
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Targeted miRNome profiling
� miFinder 384HC
� Serum & Plasma 384HC
� Cancer PathwayFinder 384HC
� Liver miFinder 384HC – New!
Sample & Assay Technologies Anatomy of a miScript miRNA PCR Array
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96-well Format: 84 miRNA + 12 controls
Cel-miR-39
miScript PCR Controls for Normalization
miRTC PPCSNORD61; SNORD68; SNORD72 SNORD95; SNORD96A; RNU6-2
RTControl
PCRControl
Spike in Control
84 miRNAs
� 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
Sample & Assay Technologies miScript miRNA PCR Arrays
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Formats built to fit your experiment and cycler
96-well 384-well
384-well (4 x 96) Rotor-Disc 100
Sample & Assay Technologies miScript miRNA PCR Arrays
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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
� Mastercycler ep realplex 2/2S/4/4S
� Mx3000p, Mx3005p, Mx4000p
� iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384� Opticon, Opticon 2, Chromo 4
� LightCycler 480
� TP-800
RotorGene Q
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 38
QIAGEN PCR Array Service Core
miScript miRNA PCR Arrays
� Total RNA Isolation: miRNeasy Kits
� Reverse Transcription: miScript II RT Kit
� qPCR: miScript miRNA PCR Arrays
� Data analysis included!
Send your samples and receive results!
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 39
Rapid Workflow = Robust, Reproducible Performance
miScript miRNA PCR Arrays
y = 1.0075x + 0.2891
R2 = 0.989
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Mean CT: Biological Replicate 2
1st Time Array User
� 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
� 1 hour� HiSpec Buffer
� 2 minutes
� 2 hours
� 15 minutes
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 40
Rapid Workflow = Robust, Reproducible Performance
miScript miRNA PCR Arrays
y = 0.9994x - 0.1945
R2 = 0.9959
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Two Operators
� Total HeLa S3 (miRNeasy)� HiSpec Buffered cDNA� miScript real-time PCR
� miFinder miScript miRNA PCR Array� Rotor-Gene Q
� 1 hour� HiSpec Buffer
� 2 minutes
� 2 hours
� 15 minutes
Sample & Assay Technologies Focused miRNA expression profiling
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FFPE samples
� One 5 µm FFPE section used per FFPE isolation� Each isolation is from a different section� 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)
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Sample & Assay Technologies Focused miRNA expression profiling (cont.)
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FFPE samples
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2-∆CT: Tumor Lung FFPE Tissue
2-∆CT: Tumor vs. Normal
� Significant differences exist between the mature miRNA expression levels of the two tissue types
� ± 2-fold [red lines] used as a cutoff for significance
Sample & Assay Technologies High Content (HC) miRNA expression profiling
Webinar 1: Meeting the Challenges of miRNA Research 43
Serum sample
2-∆CT: Cancer vs. Normal
� Workflow� 200 µl serum � 14 µl total RNA � 1.5 µl total RNA, HiSpec Buffer � Serum & Plasma 384HC miScript
miRNA PCR Array
� Significant differences exist between the mature miRNA expression levels of the two tissue types
� ± 3-fold [red lines] used as a cutoff for significance
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2-∆CT: Lung Cancer Serum
Fold-Regulation: Cancer vs. Normal
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Sample & Assay Technologies miRNome miRNA expression profiling
Webinar 1: Meeting the Challenges of miRNA Research 44
HCT 116 colorectal cancer cells
Scatter Plot Volcano Plot
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� 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
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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 PCR Kit
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Straightforward, robust, and reproducible
y = 0.9973x + 0.2058
R2 = 0.9913
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� Universal reference RNA� Two operators
� Experienced operator� First time operator
� miScript real-time PCR� miFinder miScript miRNA PCR Array
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
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∆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
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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
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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!
Webinar 1: Meeting the Challenges of miRNA Research 49
� 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
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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
Sample & Assay Technologies miScript miRNA Inhibitors
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
Sample & Assay Technologies RT2 Profiler miRNA Targets PCR Array
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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
www.qiagen.com/GeneGlobe
Sample & Assay Technologies QIAGEN’s miRNA portfolio
58
Your miRNA workflow, from sample to results!
miRNeasy Mini Kit, miRNeasy Micro Kit miScript II RT K it and PreAMP Kit HiPerFect Transfection Reagent
miRNeasy 96 Kit miScript SYBR Green PCR Kit Attractene Transfection R eagent
miRNeasy FFPE Kit miScript miRNA PCR Arrays miScript miRNA Mimics
miRNeasy Serum/Plasma Kit miScript miRNA Data Analysi s Tool miScript miRNA Inhibitors
PAXgene Tissue miRNA Kit miScript Primer Assay Custom miScript miRNA Mimics
PAXgene Blood miRNA Kit miScript Precursor Assay miSc ript Target Protector
Supplementary protocol for miRNA from Plasma and Serum
miScript PCR Starter KitmiScript miRNA Inhibitor 96 and 384 Plates and Sets
Pro
filin
g
QIAcube QIAgility Rotor-Gene Q
Webinar 1: Meeting the Challenges of miRNA Research
QIAGEN Service Core
FunctionalizationIsolationQuantification and profiling
Sample & Assay Technologies Upcoming Webinars
Webinar 1: Meeting the Challenges of miRNA Research 59
Experimental Setup and miRNA Profiling Webinars
Webinar 3: Profiling miRNA expression: on the road to biomarker development
Speaker: Jonathan Shaffer, Ph.D.
Webinar 2 : microRNA expression analysis: From experimental design to data analysis
Speaker: Jonathan Shaffer, Ph.D.
Sample & Assay Technologies Thank you for attending
Webinar 1: Meeting the Challenges of miRNA Research 60
http://www.sabiosciences.com/promotion/miscriptdemo.php
Technical Support Contact Information
� Monday through Friday
� 8:00AM to 6:00PM EST
� Direct Phone: 888-503-3187
� E-Mail: support@sabiosciences.com
Sample & Assay Technologies
Webinar 1: Meeting the Challenges of miRNA Research 61
Thank you for attending today’s webinar!
Jonathan Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.commicroRNA Technologies, R&D Americas
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