Sample & Assay Technologies

miRNA Profiling:
On the Road to Biomarker Development

Jonathan Shaffer, Ph.D.
Jonathan.Shaff...
Sample & Assay Technologies

Three part webinar series
miRNA and its role in human disease

Webinar 1 :
Speaker:

Webinar ...
Sample & Assay Technologies

Profiling miRNA expression in cells, FFPE, and serum
Agenda

Introduction to miRNA and diseas...
microRNA (miRNA) timeline

Sample & Assay Technologies

Natural phenomenon, discovery tool, potential biomarker
dsRNA, Dic...
Sample & Assay Technologies

Why quantify miRNA?

Virtually every publication includes characterization by quantification....
Sample & Assay Technologies

Canonical pathway of miRNA biogenesis

Transcribed by RNA polymerase II as
a long primary tra...
Sample & Assay Technologies

Potential events that disrupt normal miRNA activity
Disruption of miRNA–mRNA interaction

Alt...
Sample & Assay Technologies

miRNA Isolation Technologies

Isolation

Quantification
and Profiling

Functionalization

Web...
Sample & Assay Technologies

miRNeasy Kits: Mini, Micro
Flexible protocols: total RNA or enrichment of miRNA

High quality...
Sample & Assay Technologies

miRNeasy FFPE Kit
High quality total RNA from archival samples

500 million FFPE tissues are ...
Sample & Assay Technologies

miRNeasy Serum/Plasma Kit
Purification from serum, plasma, and other fluids

For purification...
Sample & Assay Technologies

miRNA in Blood
RBC, WBC, platelets, CTC, ‘other cells’, extracellular?

What is blood?
RBC, W...
Sample & Assay Technologies

Stable miRNA in circulation ~2007
An evolving story
Ago

miRNA
Exosomes &
micro vesicles1,2,3...
Sample & Assay Technologies

Plasma

Tears

Urine

Extracellular miRNAs are found in all body fluids

Breast
milk

Seminal...
Sample & Assay Technologies

miRNA in cancer diagnosis

Use of miRNA based
classifier to identify tissue
of origin for can...
Sample & Assay Technologies

miRNA Quantification Technologies

Isolation

Quantification
and Profiling

Functionalization...
Sample & Assay Technologies

miScript PCR System
Complete miRNA quantification system
1.

miScript II RT Kit
HiFlex Buffer...
Sample & Assay Technologies

miScript II RT Kit
One box with a complete miRNA quantification solution

miScript II RT Kit
...
miScript II RT Kit
Reverse transcription using HiSpec Buffer
Linearity: 2 µg to 10 pg
40
miR-16
miR-20a

35

miR-21

Mean ...
Sample & Assay Technologies

miRNA Profiling Technologies

Isolation

Quantification
and Profiling

Functionalization

Web...
Sample & Assay Technologies

miRNA expression profiling applications

Mechanisms of gene regulation
Developmental biology
...
Sample & Assay Technologies

miScript miRNA PCR Arrays
Pre-formatted solutions for miRNA profiling experiments

Pre-format...
Sample & Assay Technologies

miRNome miScript miRNA PCR Arrays
Leading coverage and validated assays

miRNome Arrays
Human...
Sample & Assay Technologies

Focused miScript miRNA PCR Arrays
Biologically relevant, intelligently designed

Focused Arra...
Sample & Assay Technologies

High Content (HC) miScript miRNA PCR Arrays
Targeted miRNome profiling
miFinder 384HC
Serum &...
Sample & Assay Technologies

Anatomy of a miScript miRNA PCR Array
96-well Format: 84 miRNA + 12 controls

84 miRNAs

Cel-...
Sample & Assay Technologies

miScript PCR Controls
Stable expression and excellent amplification efficiencies
Tissue Expre...
Sample & Assay Technologies

miScript miRNA PCR Arrays
Formats built to fit your cycler AND your experiment

96-well

384-...
Sample & Assay Technologies

miScript miRNA PCR Arrays
Compatible with a wide range of instruments

96-Well: 7000, 7300, 7...
Sample & Assay Technologies

miScript miRNA PCR Arrays
QIAGEN PCR Array Service Core

Send your samples and receive result...
Sample & Assay Technologies

miScript miRNA PCR Arrays
Rapid Workflow = Robust, Reproducible Performance
1st Time Array Us...
Sample & Assay Technologies

miScript PreAMP PCR Kit: decrease input up to 1000X
An entire miRNome from only 10 ng of tota...
Sample & Assay Technologies

miRNome miRNA expression profiling
HCT 116 colorectal cancer cells

Scatter Plot

Volcano Plo...
Sample & Assay Technologies

Focused miRNA expression profiling
FFPE samples

Reproducibility

2-∆CT: Lung Tumor vs. Norma...
Sample & Assay Technologies

miScript PreAMP: Excellent result with 250-fold less input
Limiting FFPE sample

Workflow
No ...
Sample & Assay Technologies

-80° vs. RT for 24 hrs
C

miRNA Expression Profiling from Body Fluids

Freeze-thaw cycles

We...
Sample & Assay Technologies

Workflow: Serum & plasma miRNA expression profiling

1. Collect whole blood and separate seru...
Sample & Assay Technologies

miRNA expression profiling using serum samples
High Content (HC) Array: 384 assays
2-∆CT: Can...
Sample & Assay Technologies

miScript PreAMP: Excellent result with 300-fold less input

Limiting serum sample
Reproducibi...
Sample & Assay Technologies

Workflow: Urine miRNA expression profiling
Preamplification is highly recommended

No PreAMP:...
Sample & Assay Technologies

Urine miRNA profiling: PreAMP is recommended
Results are highly reproducible

Workflow
200 µl...
Sample & Assay Technologies

Biomarker discovery workflow
Concept
Phase I (determined expressed miRNAs):
Pooled sample pro...
Sample & Assay Technologies

Biofluids: miRNA data normalization

Webinar 3: Profiling miRNA expression in cells, FFPE, an...
Sample & Assay Technologies

Data normalization
Step 1

Check reverse transcription (miRTC) and PCR (PPC) controls

Positi...
Sample & Assay Technologies

Data normalization
Step 2

Check snoRNAs/snRNA controls
Control

CT: Serum Sample A

CT: Seru...
miScript PCR Controls

Sample & Assay Technologies

Strong expression in 100% blood, low expression in serum
100% Blood:
C...
Sample & Assay Technologies

Data normalization
Step 3

Calibrate data of each plate to its RNA Recovery Controls
Can only...
Sample & Assay Technologies

Where can I find miScript miRNA PCR Arrays?
www.sabiosciences.com/mirna_pcr_array.php

miRNA ...
Sample & Assay Technologies

Where can I find miScript Primer Assays?
www.qiagen.com/GeneGlobe

Webinar 3: Profiling miRNA...
Sample & Assay Technologies

RT2 Profiler miRNA Targets PCR Array
What genes are regulated by your miRNA of interest?

Ben...
Sample & Assay Technologies

QIAGEN’s miRNA portfolio
Your miRNA workflow, from sample to results!

Quantification
and pro...
Sample & Assay Technologies

Upcoming webinars
May 2013

Sign up at http://www.SABiosciences.com/seminarlist.php
Webinar 3...
Sample & Assay Technologies

Thank you for attending

Technical Support Contact Information
Monday through Friday
8:00AM t...
Sample & Assay Technologies

Questions?
Thank you for attending today’s webinar!

Jonathan Shaffer, Ph.D.
Jonathan.Shaffer...
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Mi rna part iii_2013

  1. 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. 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. 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 3
  4. 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 4
  5. 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 5
  6. 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 6
  7. 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 7
  8. 8. Sample & Assay Technologies miRNA Isolation Technologies Isolation Quantification and Profiling Functionalization Webinar 3: Profiling miRNA expression in cells, FFPE, and serum 8
  9. 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 9
  10. 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 10
  11. 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 11
  12. 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 12
  13. 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 13
  14. 14. Sample & Assay Technologies Plasma Tears Urine Extracellular miRNAs are found in all body fluids Breast milk Seminal fluid Saliva Amniotic fluid Bronchial lavage Cerebrospinal fluid Pleural fluid Peritoneal fluid miR-335* miR-335* miR-515-3p miR-335* miR-518e miR-335* miR-518e miR-515-3p miR-335* miR-515-3p miR-515-3p miR-325 miR-515-3p miR-335* miR-26a-2* miR-590-3p miR-892a miR-335* miR-335* miR-515-3p miR-892a miR-892a miR-377* miR-137 miR-892a miR-181d miR-588 miR-515-3p miR-302c miR-509-5p miR-892a miR-223* miR-518e miR-586 miR-509-5p miR-509-5p miR-509-5p miR-873 miR-545* miR-515-3p miR-616* miR-873 miR-616* miR-134 miR-518e miR-590-3p miR-223* miR-524-5p miR-590-5p miR-27a miR-452 miR-545* miR-509-5p miR-873 miR-509-5p let-7i miR-873 miR-873 miR-137 miR-197 miR-518e miR-892a miR-302d miR-223* miR-335* miR-223* miR-539 miR-892a miR-302d miR-26a-1* miR-218 miR-509-5p miR-671-5p miR-223* miR-302d miR-302d miR-515-5p miR-616* miR-223* miR-616* miR-595 miR-515-5p miR-492 miR-515-5p miR-25* miR-616* miR-134 miR-616* miR-302d miR-302d miR-134 miR-580 miR-137 miR-483-5p miR-137 miR-134 miR-483-5p miR-509-5p miR-302d miR-589 miR-590-5p miR-923 miR-130a miR-335* miR-923 miR-593* miR-483-5p miR-134 miR-377* miR-873 miR-556-3p miR-195 miR-483-5p miR-515-3p miR-617 miR-873 miR-590-3p miR-923 miR-518e miR-589 miR-483-5p miR-151-3p miR-130a miR-101* miR-513c miR-410 miR-92b miR-873 miR-518e miR-923 miR-518e miR-923 miR-548b-3p miR-675 miR-325 miR-671-5p miR-524-5p miR-302d miR-410 miR-548b-3p miR-377* miR-556-3p miR-374a miR-192 miR-29b miR-589 miR-490-5p miR-20b let-7a miR-509-5p miR-1225-3p miR-589 miR-652 miR-598 miR-151-5p miR-616* miR-556-3p miR-367 miR-181d miR-580 miR-548d-5p miR-325 miR-556-3p miR-590-5p miR-548b-3p miR-598 miR-410 miR-545* miR-181b miR-1 miR-616* miR-223* miR-589 miR-652 miR-923 miR-1238 mir-187 miR-101* miR-377* miR-598 miR-671-5p miR-134 miR-616* miR-92b miR-498 miR-371-3p miR-92b miR-873 miR-134 let-7i miR-515-5p miR-509-5p miR-25* miR-148b* miR-371-3p miR-767-3p miR-890 miR-498 miR-218 miR-487b miR-890 miR-578 miR-515-3p let-7i miR-590-5p miR-324-3p miR-505 miR-498 miR-937 miR-923 miR-483-5p miR-505 miR-487b miR-892a miR-410 miR-302d miR-885-5p miR-151-5p miR-483-5p miR-377* Weber et al. (2010) Clinical Chem. 56: 1733-1741. miRNeasy, miScript PCR System Webinar 3: Profiling miRNA expression in cells, FFPE, and serum 14
  15. 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. 16. Sample & Assay Technologies miRNA Quantification Technologies Isolation Quantification and Profiling Functionalization Webinar 3: Profiling miRNA expression in cells, FFPE, and serum 16
  17. 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 17
  18. 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 18
  19. 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 2 3 4 5 Log copy number of miRNA using HiSpec Buffer 6 19
  20. 20. Sample & Assay Technologies miRNA Profiling Technologies Isolation Quantification and Profiling Functionalization Webinar 3: Profiling miRNA expression in cells, FFPE, and serum 20
  21. 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 21
  22. 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 22
  23. 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 23
  24. 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 24
  25. 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 25
  26. 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 26
  27. 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 27
  28. 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 28
  29. 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 29
  30. 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 30
  31. 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 31
  32. 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 32
  33. 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 33
  34. 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 34
  35. 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 35
  36. 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 36
  37. 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 37
  38. 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 38
  39. 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 39
  40. 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 40
  41. 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 41
  42. 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 42
  43. 43. Sample & Assay Technologies Biofluids: miRNA data normalization Webinar 3: Profiling miRNA expression in cells, FFPE, and serum 43
  44. 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 44
  45. 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 45
  46. 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 46
  47. 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 47
  48. 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 48
  49. 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 49
  50. 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 50
  51. 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 51
  52. 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 52
  53. 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 53
  54. 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 Webinar 3: Profiling miRNA expression in cells, FFPE, and serum 54

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