Sample & Assay Technologies

Advanced miRNA Expression Analysis:
From Experimental Design through Data Analysis

Jonathan ...
Sample & Assay Technologies

Three part webinar series
miRNA and its role in human disease

Webinar 1 :
Speaker:

Webinar ...
Sample & Assay Technologies

Advanced miRNA Expression Analysis
Agenda

Overview
miRNA Background
miRNA expression profili...
Sample & Assay Technologies

Canonical pathway of miRNA biogenesis

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

Why quantify miRNAs?

HighWire + PubMed Publications

miRBase Entries

Virtually every public...
Sample & Assay Technologies

miRNA expression profiling applications

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

miRNA quantification approaches

Northern blotting
Deep sequencing
Microarrays
Real-time PCR ...
Sample & Assay Technologies

miRNA quantification by real-time PCR

Real-time PCR quantification of miRNAs
Has been the go...
Sample & Assay Technologies

miRNA expression profiling using real-time PCR
Key considerations

Scientific question
Well-d...
Sample & Assay Technologies

miScript PCR System
Fully integrated, complete miRNA quantification system
1.

miScript II RT...
Sample & Assay Technologies

miScript II RT Kit
A complete miRNA quantification solution

miScript II RT Kit

Mature miRNA...
Sample & Assay Technologies

miScript II RT Kit
Reverse transcription using HiSpec Buffer

Webinar 2: Advanced miRNA Expre...
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-...
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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...
miScript miRNA PCR Arrays
Rapid Workflow = Robust, Reproducible Performance
1st Time Array User
1 hour
HiSpec Buffer

2 mi...
Sample & Assay Technologies

Real-time PCR data analysis

Absolute quantification
Absolute input copies, based on a standa...
Sample & Assay Technologies

Data analysis workflow

Steps 1 & 2:
Set Baseline and Threshold to determine CT values

Step ...
Sample & Assay Technologies

Steps 1 & 2
Set Baseline and Threshold to determine CT values

Baseline
Definition: Noise lev...
Sample & Assay Technologies

Steps 1 & 2
Set Baseline and Threshold to determine CT values

Threshold
Purpose: Used to det...
Sample & Assay Technologies

Setting the Baseline and Threshold
Applied Biosystems® 7900HT

Baseline: 3 to 15

Threshold: ...
Sample & Assay Technologies

Setting the Baseline and Threshold
QIAGEN Rotor-Gene® Q

Baseline

Threshold: 0.02

Always se...
Sample & Assay Technologies

miScript miRNA PCR Arrays on Roche® LightCycler® 480
Use the Second Derivative Maximum analys...
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Step 3: Export CT values

Normal Lung

40

36

32

32

28

28
CT Value

36

CT Value

Lung Tu...
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Step 4: Analyze data
∆∆CT method of relative quantification

Normal (N) Lung Total RNA
N cDNA...
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Step 4: Analyze data (cont.)
∆∆CT method of relative quantification

Normal (N) Lung

Lung Tu...
Sample & Assay Technologies

Step 4: Analyze data (cont.)
∆∆CT method of relative quantification
Fold-Regulation: Tumor vs...
Sample & Assay Technologies

miScript’s straightforward, data analysis workflow
Incorporating the free miScript miRNA PCR ...
Sample & Assay Technologies

Steps 1 & 2
Set Baseline and Threshold to determine CT values

Baseline
Definition: Noise lev...
Sample & Assay Technologies

Steps 3 & 4
Export CT values and access free analysis tools

Export CT values according to th...
Sample & Assay Technologies

Step 5: Automatic data analysis
miScript miRNA PCR Array Data Analysis Tools

Web-based softw...
Sample & Assay Technologies

miScript miRNA PCR Array Data Analysis
Step 5A

Website dedicated for array data analysis
htt...
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miScript miRNA PCR Array Data Analysis
Step 5B

Readout Tab

Basic Setup

3. Click Update All...
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miScript miRNA PCR Array Data Analysis
Step 5C
Readout Tab

View Housekeeping Genes

Choose ‘...
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miScript miRNA PCR Array Data Analysis
Step 5D

Analysis Result Tab: this tab provides an ove...
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miScript miRNA PCR Array Data Analysis
Step 5E

Scatter Plot, Volcano Plot, Clustergram, and ...
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miScript miRNA PCR Array Data Analysis
Step 5F

Export Data Tab: When clicked, results calcul...
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Serum Sample analysis using the miScript PCR System

Webinar 2: Advanced miRNA Expression Ana...
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Workflow: Serum & plasma miRNA expression profiling

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

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

Special data analysis case
Serum & Plasma Samples

Serum or plasma total RNA samples: The sno...
Sample & Assay Technologies

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

84 miRNAs

Cel-...
Serum or plasma sample data normalization

Sample & Assay Technologies

Calibrate samples using cel-miR-39 CT mean
Uncalib...
Sample & Assay Technologies

Serum or plasma sample data normalization
Option 1: CT values normalized to CT mean of expres...
Sample & Assay Technologies

Serum or plasma sample data normalization
Option 2: CT values normalized to CT mean of invari...
Sample & Assay Technologies

Serum or plasma sample data normalization
Comparison of normalization methods

Option 1:
Comm...
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Serum or plasma sample data normalization
miRNA expression profiling
2-∆CT: Breast Cancer vs....
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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 2: Advanced miRNA ...
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QIAGEN’s miRNA portfolio
Your miRNA workflow, from sample to results!

Quantification
and pro...
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Upcoming webinars
Experimental Setup and miRNA Profiling webinars

Webinar 3: Profiling miRNA...
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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 ii_2013

  1. 1. Sample & Assay Technologies Advanced miRNA Expression Analysis: From Experimental Design through Data Analysis Jonathan Shaffer, Ph.D. Jonathan.Shaffer@qiagen.com microRNA Technologies, R&D Americas The products described in this webinar are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of disease.
  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 2: Advanced miRNA Expression Analysis 2
  3. 3. Sample & Assay Technologies Advanced miRNA Expression Analysis Agenda Overview miRNA Background miRNA expression profiling using a miScript miRNA PCR Array How to calculate fold-change using the ∆∆CT method of relative quantification Setting the Baseline and Threshold Data analysis example 1: Basic Experiment Using the free miScript miRNA PCR Array data analysis tools Data analysis example 2: Serum miRNA Experiment Summary of QIAGEN’s miRNA detection portfolio Current Promotion Webinar 2: Advanced miRNA Expression Analysis 3
  4. 4. 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 2: Advanced miRNA Expression Analysis 4
  5. 5. Sample & Assay Technologies Why quantify miRNAs? HighWire + PubMed Publications miRBase Entries 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 2: Advanced miRNA Expression Analysis 5
  6. 6. 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 2: Advanced miRNA Expression Analysis 6
  7. 7. Sample & Assay Technologies miRNA quantification approaches Northern blotting Deep sequencing Microarrays Real-time PCR based approaches Webinar 2: Advanced miRNA Expression Analysis 7
  8. 8. Sample & Assay Technologies miRNA quantification by real-time PCR Real-time PCR quantification of miRNAs Has been the gold standard for gene quantification Is the method of choice to confirm next-generation sequencing and microarray results Simple and easy to carry out High sensitivity, specificity High throughput compatible, automatable Needs very low amounts of template Webinar 2: Advanced miRNA Expression Analysis 8
  9. 9. Sample & Assay Technologies miRNA expression profiling using real-time PCR Key considerations Scientific question Well-defined and testable Experimental sample set Statistically meaningful number of replicates (biological replicates) A minimum of three replicates recommended Additional replicates may enhance statistical power Inclusion of proper controls Experimental testing platform Simple, straightforward workflow High sensitivity and specificity When profiling expression: a variety of PCR arrays to meet your experimental needs Easy and simple data analysis tools Customizable solutions Availability of companion research tools Webinar 2: Advanced miRNA Expression Analysis 9
  10. 10. Sample & Assay Technologies miScript PCR System Fully integrated, complete miRNA quantification system 1. miScript II RT Kit HiFlex Buffer: Unparalleled flexibility for quantification of miRNA and mRNA 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 Webinar 2: Advanced miRNA Expression Analysis 10
  11. 11. Sample & Assay Technologies miScript II RT Kit A complete miRNA quantification solution miScript II RT Kit Mature miRNA quantification and profiling? Biogenesis studies? 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 2: Advanced miRNA Expression Analysis 11
  12. 12. Sample & Assay Technologies miScript II RT Kit Reverse transcription using HiSpec Buffer Webinar 2: Advanced miRNA Expression Analysis 12
  13. 13. 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 Webinar 2: Advanced miRNA Expression Analysis miRBase V17 and V18 assays are available! Contact us if you are interest in a different species! 13
  14. 14. 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 Webinar 2: Advanced miRNA Expression Analysis 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! 14
  15. 15. 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 2: Advanced miRNA Expression Analysis 15
  16. 16. 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 2: Advanced miRNA Expression Analysis 16
  17. 17. 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 2: Advanced miRNA Expression Analysis 17
  18. 18. 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 2: Advanced miRNA Expression Analysis 18
  19. 19. 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 2: Advanced miRNA Expression Analysis 19
  20. 20. 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 2: Advanced miRNA Expression Analysis 20
  21. 21. miScript miRNA PCR Arrays Rapid Workflow = Robust, Reproducible Performance 1st Time Array User 1 hour HiSpec Buffer 2 minutes 30 Mean CT: Biological Replicate 2 Sample & Assay Technologies 25 20 y = 1.0075x + 0.2891 2 hours 2 R = 0.989 15 15 20 25 30 Mean CT: Biological Replicate 1 15 minutes Webinar 2: Advanced miRNA Expression Analysis 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 21
  22. 22. Sample & Assay Technologies Real-time PCR data analysis Absolute quantification Absolute input copies, based on a standard curve Relative quantification Comparative CT method: also known as the 2-∆∆CT method Selection of internal control Selection of calibrator (e.g. untreated control or normal sample) Assumes that the PCR efficiency of the target gene is similar to the internal control gene (and that the efficiency of the PCR is close to 100%) Fold change = 2-∆∆CT CT = 23.8 – ∆CT = CTGene - CTNormalizer – ∆∆CT = ∆CT (sample 2) – ∆CT (sample 1) where sample 1 is the control sample and sample 2 is the experimental sample (1) Schmittgen TD, Livak KJ.(2008):Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc.;3(6):1101-8 (2) Livak, KJ, and Schmittgen, TD.(2001): Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-∆∆CT Method METHODS 25, 402–408 (3) www.Gene-Quantification.info Webinar 2: Advanced miRNA Expression Analysis 22
  23. 23. Sample & Assay Technologies Data analysis workflow Steps 1 & 2: Set Baseline and Threshold to determine CT values Step 3: Export CT values Step 4: Analyze data using ∆∆CT method of relative quantification Webinar 2: Advanced miRNA Expression Analysis 23
  24. 24. Sample & Assay Technologies Steps 1 & 2 Set Baseline and Threshold to determine CT values Baseline Definition: Noise level in early cycles where there is no detectable increase in fluorescence due to PCR products. How to Set: – – – – Observe amplification plot using the “Linear View” Determine the earliest visible amplification Set the baseline from cycle 2 to 2 cycles before the earliest visible amplification Note: The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used Webinar 2: Advanced miRNA Expression Analysis 24
  25. 25. Sample & Assay Technologies Steps 1 & 2 Set Baseline and Threshold to determine CT values Threshold Purpose: Used to determine the CT (threshold cycle) value. The point at which the amplification curve intersects with the threshold line is called the CT. How to Set: – Observe amplification plot using the “Log View” – Place the threshold in the lower half of the log-linear range of the amplification plot, above the background signal – Note: Never set the threshold in the plateau phase Webinar 2: Advanced miRNA Expression Analysis 25
  26. 26. Sample & Assay Technologies Setting the Baseline and Threshold Applied Biosystems® 7900HT Baseline: 3 to 15 Threshold: 0.2 Baseline: From cycle 2 (or 3) to 2 cycles before the earliest visible amplification. Threshold: Place in the lower half of the log-linear range of the amplification plot, above the background signal. Webinar 2: Advanced miRNA Expression Analysis 26
  27. 27. Sample & Assay Technologies Setting the Baseline and Threshold QIAGEN Rotor-Gene® Q Baseline Threshold: 0.02 Always select “Dynamic Tube” Potentially select “Slope Correct” and/or “Ignore First” Webinar 2: Advanced miRNA Expression Analysis 27
  28. 28. Sample & Assay Technologies miScript miRNA PCR Arrays on Roche® LightCycler® 480 Use the Second Derivative Maximum analysis method Select Second Derivative Maximum analysis method Webinar 2: Advanced miRNA Expression Analysis Obtain CT Values 28
  29. 29. Sample & Assay Technologies Step 3: Export CT values Normal Lung 40 36 32 32 28 28 CT Value 36 CT Value Lung Tumor 40 24 20 24 20 16 16 12 FFPE Isolation 1 12 FFPE Isolation 2 8 FFPE Isolation 1 FFPE Isolation 2 8 FFPE Isolation 3 FFPE Isolation 3 4 4 1 7 13 19 25 31 37 43 49 55 61 67 73 1 7 13 19 25 31 37 43 49 55 61 67 73 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) Webinar 2: Advanced miRNA Expression Analysis 29
  30. 30. Sample & Assay Technologies Step 4: Analyze data ∆∆CT method of relative quantification Normal (N) Lung Total RNA N cDNA (Iso. 1) N cDNA (Iso. 2) Lung Tumor (T) total RNA N cDNA (Iso. 3) Exported CT values T cDNA (Iso. 1) Calculate ∆CT for each miRNA on each array ∆CT = CTmiRNA – AVG CTSN1/2/3/4/5/6 T cDNA (Iso. 2) T cDNA (Iso. 3) Exported CT values ∆CT = CTmiRNA – AVG CTSN1/2/3/4/5/6 Tip for choosing an appropriate snoRNA/snRNA controls for normalization Make sure that the selected controls are not influenced by the experimental conditions Webinar 2: Advanced miRNA Expression Analysis 30
  31. 31. Sample & Assay Technologies Step 4: Analyze data (cont.) ∆∆CT method of relative quantification Normal (N) Lung Lung Tumor (T) Calculate ∆CT for each miRNA on each array ∆CT ∆CT ∆CT ∆CT ∆CT ∆CT Calculate Average ∆CT for each miRNA within group (N or T) ∆CT + ∆CT + ∆CT ∆CT + ∆CT + ∆CT 3 3 Calculate ∆∆CT for each miRNA between groups (T – N) ∆∆CT = Avg. ∆CT (T) – Avg. ∆CT (N) Calculate fold-change for each miRNA (T vs. N) 2-(∆∆CT) Webinar 2: Advanced miRNA Expression Analysis 31
  32. 32. Sample & Assay Technologies Step 4: Analyze data (cont.) ∆∆CT method of relative quantification Fold-Regulation: Tumor vs. Normal Log2 Fold-Regulation (Tumor vs. Normal) 14 10 6 2 -2 -6 -10 -14 Significant differences exist between the mature miRNA expression levels of the two tissue types Webinar 2: Advanced miRNA Expression Analysis 32
  33. 33. Sample & Assay Technologies miScript’s straightforward, data analysis workflow Incorporating the free miScript miRNA PCR Array Data Analysis Software Steps 1 & 2: Set Baseline and Threshold to determine CT values Step 3: Export CT values Step 4: Access the free data analysis tools at http://pcrdataanalysis.sabiosciences.com/mirna Step 5 & on: Automatic data using ∆∆CT method of relative quantification Webinar 2: Advanced miRNA Expression Analysis 33
  34. 34. Sample & Assay Technologies Steps 1 & 2 Set Baseline and Threshold to determine CT values Baseline Definition: Noise level in early cycles where there is no detectable increase in fluorescence due to PCR products. How to Set: – – – – Observe amplification plot using the “Linear View” Determine the earliest visible amplification Set the baseline from cycle 2 to 2 cycles before the earliest visible amplification Note: The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used Threshold Purpose: Used to determine the CT (threshold cycle) value. The point at which the amplification curve intersects with the threshold line is called the CT. How to Set: – Observe amplification plot using the “Log View” – Place the threshold in the lower half of the log-linear range of the amplification plot, above the background signal – Note: Never set the threshold in the plateau phase Important: Ensure that baseline and threshold settings are the same across all PCR runs in the same analysis to allow comparison of results. Webinar 2: Advanced miRNA Expression Analysis 34
  35. 35. Sample & Assay Technologies Steps 3 & 4 Export CT values and access free analysis tools Export CT values according to the manual supplied with the real-time PCR instrument Access the free miScript miRNA PCR Array Data Analysis Tools Website: http://sabiosciences.com/mirnaArrayDataAnalysis.php – Web-based – Excel-based Webinar 2: Advanced miRNA Expression Analysis 35
  36. 36. Sample & Assay Technologies Step 5: Automatic data analysis miScript miRNA PCR Array Data Analysis Tools Web-based software No installation needed Tailored for each array Raw CT values to fold-change results Using ∆∆CT Method Multiple Analysis Formats Scatter Plot Volcano Plot Multi-Group Plot Clustergram Downloadable Excel analysis templates are also available Webinar 2: Advanced miRNA Expression Analysis 36
  37. 37. Sample & Assay Technologies miScript miRNA PCR Array Data Analysis Step 5A Website dedicated for array data analysis http://pcrdataanalysis.sabiosciences.com/mirna 1. Choose Catalog Number 2. Upload exported CT values 3. Click Upload Upload Readout Tab This tab includes: Uploading instructions Data normalization instructions Instructions that walk you through the data analysis (right side of ‘Upload Readout’ Tab – not shown on this image) You can also Take a Test Run or Play Movie Guide for help with using the software What should you do at this page? 1. 2. 3. Choose array catalog number from ‘Catalog Number’ dropdown menu Upload exported CT values from computer Click Upload Webinar 2: Advanced miRNA Expression Analysis 37
  38. 38. Sample & Assay Technologies miScript miRNA PCR Array Data Analysis Step 5B Readout Tab Basic Setup 3. Click Update All Changes 1. Choose ‘Control Genes’ This tab includes: All miRNAs and controls found on chosen array Column where Normalization RNA (control gene) can be selected All CT data uploaded to software 2. Designate CT values column groups What should you do at this page? 1. 2. 3. Click boxes next to desired ‘Control Genes’ (miScript PCR Controls are pre-selected) Designate columns of CT values as Control, Group 1, Group 2, etc. Click ‘Update All Changes’ Webinar 2: Advanced miRNA Expression Analysis 38
  39. 39. Sample & Assay Technologies miScript miRNA PCR Array Data Analysis Step 5C Readout Tab View Housekeeping Genes Choose ‘Normalized By’ Readout Tab View Housekeeping Genes, Data Overview, & Data QC View Housekeeping Genes Tab Selected ‘Control Genes’ can be visualized and method of normalization can be chosen Data Overview Tab This tab provides an overview of all ∆∆CT calculations performed by the software Readout Tab Data QC Data QC Tab This tab provides an overview of QC data associated with the miRTC (reverse transcription control) and PPC (positive PCR control) Webinar 2: Advanced miRNA Expression Analysis 39
  40. 40. Sample & Assay Technologies miScript miRNA PCR Array Data Analysis Step 5D Analysis Result Tab: this tab provides an overview of all ∆∆CT related calculations and provides a guide for you regarding the trust that you should place in your data (see red arrow) Comments on data quality Webinar 2: Advanced miRNA Expression Analysis 40
  41. 41. Sample & Assay Technologies miScript miRNA PCR Array Data Analysis Step 5E Scatter Plot, Volcano Plot, Clustergram, and Multigroup Plot Tabs: When clicked, these tabs provide various statistical outputs that will open as new windows. The scatter plot is included as an example. Scatter Plot Webinar 2: Advanced miRNA Expression Analysis 41
  42. 42. Sample & Assay Technologies miScript miRNA PCR Array Data Analysis Step 5F Export Data Tab: When clicked, results calculated by the miScript miRNA PCR Array Data Analysis software can be exported to Microsoft Excel. Webinar 2: Advanced miRNA Expression Analysis 42
  43. 43. Sample & Assay Technologies Serum Sample analysis using the miScript PCR System Webinar 2: Advanced miRNA Expression Analysis 43
  44. 44. 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 2: Advanced miRNA Expression Analysis 44
  45. 45. 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 2: Advanced miRNA Expression Analysis 45
  46. 46. Sample & Assay Technologies Special data analysis case Serum & Plasma Samples Serum or plasma total RNA samples: The snoRNA/snRNA panel of targets does not exhibit robust expression and therefore should not be selected as Normalization Controls. Typical CT Values for miScript PCR Controls in Serum Samples Control Serum Sample 1 Serum Sample 2 Serum Sample 3 SNORD61 36.3 34.3 35.8 SNORD68 34.6 35.0 35.3 SNORD72 35.0 35.0 35.0 SNORD95 31.1 39.3 33.5 SNORD96A 33.6 34.5 35.4 RNU6-2 37.9 39.1 35.0 Step 1: Calibrate samples using cel-miR-39 CT mean Step 2: Normalize serum or plasma sample data Option 1: Normalize CT values to CT mean of all commonly expressed miRNAs Option 2: Normalize CT values to CT mean of invariant miRNAs Webinar 2: Advanced miRNA Expression Analysis 46
  47. 47. 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 2: Advanced miRNA Expression Analysis 47
  48. 48. Serum or plasma sample data normalization Sample & Assay Technologies Calibrate samples using cel-miR-39 CT mean Uncalibrated Assay Sample 1 Sample 2 hsa-miR-16 16.0 19.0 hsa-miR-21 20.0 24.0 hsa-miR-192 23.0 26.0 hsa-miR-103 23.0 23.0 hsa-miR-25 22.0 25.0 cel-miR-39 18.0 21.0 Compared to sample 1, all assays in sample 2 appear to have delayed CT values Compared to sample 1, cel-miR-39 in sample 2 also has a delayed CT value Conclusion: calibrate samples (cel-miR-39 CT values indicate a differential recovery) Calibrated (Sample 1 CT values +3) Assay Sample 1 Sample 2 hsa-miR-16 19.0 19.0 hsa-miR-21 23.0 24.0 hsa-miR-192 26.0 26.0 hsa-miR-103 26.0 23.0 hsa-miR-25 25.0 25.0 cel-miR-39 21.0 21.0 Webinar 2: Advanced miRNA Expression Analysis 48
  49. 49. Sample & Assay Technologies Serum or plasma sample data normalization Option 1: CT values normalized to CT mean of expressed miRNAs Calculate the CT mean for commonly expressed miRNAs Those miRNAs with CT values < 30 (or 32, or 35) in all assessed samples 12 Fold-Regulation 8 4 0 -4 -8 Webinar 2: Advanced miRNA Expression Analysis 49
  50. 50. Sample & Assay Technologies Serum or plasma sample data normalization Option 2: CT values normalized to CT mean of invariant miRNAs Calculate the CT mean for invariant miRNAs Choose at least 4 to 6 miRNAs that exhibit little CT variation 12 Commonly Expressed miRNAs hsa-miR-92a hsa-let-7c hsa-miR-93 hsa-miR-21 hsa-miR-103a hsa-miR-22 hsa-miR-126 hsa-miR-23a hsa-miR-145 hsa-miR-24 hsa-miR-146a hsa-miR-25 hsa-miR-191 hsa-miR-26a hsa-miR-222 hsa-miR-26b 8 Fold-Regulation hsa-let-7a hsa-miR-423-5p 4 0 -4 -8 Webinar 2: Advanced miRNA Expression Analysis 50
  51. 51. Sample & Assay Technologies Serum or plasma sample data normalization Comparison of normalization methods Option 1: Commonly Expressed miRNAs Option 2: Invariant Panel of miRNAs (miRNome, 384HC) (small panel screening) 8 Fold-Regulation 12 8 Fold-Regulation 12 4 0 4 0 -4 -4 -8 -8 Note 1: In this example, fold-regulation looks highly similar, irrespective of the chosen normalization method. This is correct, as your results should be independent of the chosen normalization method. Note 2: For small panel screening, do not use a CT mean of all miRNAs, as this array is biased (miRNA assays included on this array are not random) Webinar 2: Advanced miRNA Expression Analysis 51
  52. 52. Sample & Assay Technologies Serum or plasma sample data normalization miRNA expression profiling 2-∆CT: Breast Cancer vs. Normal Normal Serum 1.E+01 30 26 22 Serum Isolation 1 18 Serum Isolation 2 Serum Isolation 3 1.E-01 1.E-02 miR-34a 1.E-03 1.E-04 1.E-05 1.E+01 200 µl serum 50 µl total RNA 5 µl total RNA, HiSpec Buffer miFinder miScript miRNA PCR Array 1.E+00 Workflow -∆CT 1.E-01 2 1.E-02 1.E-03 miRNA 1.E-04 6 11 16 21 26 31 36 41 46 51 56 61 1.E-05 14 1 1.E+00 2 CT Value 34 -∆CT : Breast Cancer Serum 38 : Normal Serum one-half of cDNA used for 96-well High reproducibility can be achieved using the miRNeasy Supplementary Protocol Each isolation was from a different normal serum donor Significant differences exist between the mature miRNA expression levels of the two tissue types ± 2-fold [red lines] used as a cutoff for significance Webinar 2: Advanced miRNA Expression Analysis 52
  53. 53. 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 2: Advanced miRNA Expression Analysis 53
  54. 54. Sample & Assay Technologies Where can I find miScript Primer Assays? www.qiagen.com/GeneGlobe Webinar 2: Advanced miRNA Expression Analysis 54
  55. 55. 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 Webinar 2: Advanced miRNA Expression Analysis QIAgility Rotor-Gene Q 55
  56. 56. Sample & Assay Technologies Upcoming webinars Experimental Setup and miRNA Profiling webinars Webinar 3: Profiling miRNA expression: on the road to biomarker development Date: April 24, 2013, 9:30 am EDT Speaker: Jonathan Shaffer, Ph.D. Webinar 2: Advanced miRNA Expression Analysis 56
  57. 57. 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 57
  58. 58. 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 2: Advanced miRNA Expression Analysis 58

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