Ffpe pcr array

RT2 Profiler PCR Arrays
FFPE & Small Sample Gene Expression Analysis

Shankar Sellappan, Ph.D.
Global Product Manager
Sample & Assay Technologies

Welcome to SABiosciences: Who We Are

• SABiosciences is now a

Company

Central Dogma of Molecular Biology

Transcription

DNA
Genome
10,000 20,000 Genes

Translation

mRNA
Transcriptome

Protein
Proteome
~ 50,000 proteins


Gene Expression (mRNA) Regulates Biology

Biological Processes, such as Apoptosis, Cell
Cycle, Inflammation, Signal Transduction, etc.,
can be identified by specific patterns of gene
expression (pathway-focused).


Differential Gene Expression Analysis

Q: How to Assess the Expression of
Different mRNAs in a Sample and
Compare it across multiple samples?

A:


PCR Array – Sample Image


Table of Contents

1. Introduction to the PCR Arrays
2. How PCR Arrays Work with PreAMP
3. PreAMP-PCR Array Performance Data


PCR Arrays for All Biomedical Researchers
Cancer and Apoptosis

Cytokines & Inflammation

Development & Stem Cells

Apoptosis

Inflammatory Cytokines

Stem Cells

Cell Cycle

Th17 for Inflammation

WNT Signaling / Notch Signaling

Human miRNA Array (NEW!)

Common Cytokines / Chemokines

Terminal Differentiation Markers

Breast Cancer & Estrogen Receptor

Inflammasomes - NEW!

TGFβ / BMP Signaling

Tumor Metastasis

NF-kB Signaling Pathway

Endothelial Cell Biology

Epithelial-to-Mesenchymal Transition

Th1-Th2-Th3

Osteogenesis

Angiogenesis

TNF Ligands

Growth Factors

Cancer Drug Resistance

Toll-like Receptors

ECM & Adhesion

Signal Transduction

Toxicology & Drug Metabolism

Neuroscience

Signal Transduction PathwayFinder

Drug Metabolism

Neuroscience Ion Channels

NFkB Signaling

Drug Phase I Enzymes

Neurotransmitter Receptors

Jak / Stat Signaling

Drug Transporters

Neurotrophins & Receptors

DNA Damage Signaling

Oxidative Stress

Neurogenesis and Neural Stem Cell

Insulin Signaling

Stress & Toxicity

MAP Kinase Signaling

Other Diseases

Custom PCR Arrays (H/M/R/Q/D/F)

cAMP / Calcium Signaling

Atherosclerosis

96-Well, 384-Well Plate

p53 Signaling

Diabetes

100-Well Disc, 96x96 Chip

** Over 100 Pathway-Powered PCR Arrays Available**

Primer Assays – Laboratory Verification Data

Melting/Dissociation Curve
• Single Peak

Amplification Plot
• PCR Efficiency


How Genes on PCR Arrays Are Selected
• Biologically relevant gene content
• Not simply biochemical pathways or kinase
cascades
• Published association with the biological or
disease pathway gathered from overlapping
sources, including:
• Multiple Publicly Accessible Databases
• Text Mining Relevant Literature
• Technically relevant gene content
• Use genes that are regulated at the mRNA
level
• Specific feedback from thought leaders

Genes in Signaling Pathways

Genes with High Relevance


Relevant Pathway Content on PCR Arrays


Complete PCR Array System – Bench Verified
• The entire system has been optimized and bench-verified and
bench
we guarantee their performance when used together!
• RNA isolation kits (verified)
• RT2 Profiler PCR Arrays - Bench verified
• RT2 1st Strand cDNA synthesis kit
–
–
–
–

Efficient genomic DNA removal step
Built-in reverse transcription control to monitor sample quality
A hybrid method of random primers and Oligo dT
Fast (20 minutes); High-throughput available

• RT2 SYBR Green Master Mixes
– Unique buffer system to ensure PCR specificity and efficiency

• Free PCR Array Data Analysis software
– Web & Excel based

Publish in High Impact Factor Journals


Principles of qRT-PCR in PCR Arrays

Real-Time PCR
• Amplify & simultaneously quantify target DNA
Reverse Transcription Real-Time PCR
• Amplify & simultaneously quantify messenger RNA (mRNA)

Ct Values
• Threshold Cycle

Anatomy of a PCR Array

• 84 Pathway-Specific
Genes of Interest
• 5 Housekeeping Genes
• Genomic DNA
Contamination Control
• Reverse Transcription
Controls (RTC) n=3
• Positive PCR Controls
(PPC) n=3
Supported PCR Array Formats
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•
•
•

96-Well Plate
384-Well Plate
100-Well Disc
96x96 Chip

Compatible Instrumentation
96-, 100-, 384-, and 96x96-Well Formats

•
•
•

96-Well Blocks: 7000, 7300, 7500, 7700,
ViiA 7
FAST 96-Well Blocks: 7500, 7900HT, Step
One Plus, ViiA 7
FAST 384-Well Block: 7900HT, ViiA 7

•
•

iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384
Opticon, Opticon 2, Chromo 4

•

Mastercycler ep realplex 2/2S/4/4S

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BioMark

•

Rotor Gene Q

•

LightCycler 480

•

Mx3000p, Mx3005p, Mx4000p

•

TP-800
• New Instrument

PCR Array Service Core

• Set-up protocol available


Plate Formats & Layouts
96-well, 100-well, 384-well (4x96), 384-well HT


PCR Arrays Compatible with ALL Types of Samples

• Cultured cells
• QIAGEN RNeasy Kits with On-Column DNase Treatment

• Tissues
• QIAZOL followed by RNeasy Kits with On-Column DNase Treatment

• Blood samples
• QIAGEN PaxGene Blood RNA kit

• Small samples
• QIAGEN RNeasy Micro Kit & RT2 PreAMP system
• Fine needle biopsy (FNB) samples
• Laser captured microdissection (LCM) samples
• Stem cell clusters or embryoid bodies
• Fluorescence-Activated Cell Sorter (FACS) generated cells

• FFPE (Formalin-fixed Paraffin-embedded)
• QIAGEN RNeasy FFPE Kit & RT2 PreAMP system

RT2 Profiler™ PCR Array Data Analysis

• FREE Complete & Easy Data Analysis with Web-Based Software
• From Raw Ct Values to Fold Change Results in Multiple Analysis Formats
Volcano Plot

Scatter Plot

Heat Map

3-D Histogram

Register for PCR Array Data Analysis webinar to learn more!
Bookmark:http://www.SABiosciences.com/pcr/arrayanalysis.php


Table of Contents

1.

Introduction to the PCR Arrays

2.

How PCR Arrays Work
With PreAMP Technology

3. PreAMP-PCR Array Performance Data


How Pathway-Powered PCR Arrays Work
Control & Experimental Cells or Tissues
1. Isolate Total RNA

25 ng – 5 ug

2. Reverse Transcription: RT2 First
Strand cDNA Synthesis
3. Add RT2 qPCR SYBR Green Master Mix & cDNA
Cocktail & Load PCR Array
4. Real-Time PCR
(40 cycles)
5. Data Analysis: Determine Fold Changes (∆∆ Ct Method)
∆∆


How RT2 PCR Arrays Work: 25 ng – 5 µg RNA

RNA (25 ng – 5 ug)
gDNA Elimination Step
~45 min
First Strand Synthesis
1st strand cDNA

+ SYBR Green Master Mix
2 hours

Real-time PCR Detection of 89
Gene-specific Amplification on
RT2 Profiler PCR Array


PCR Arrays: Sensitivity of Detection
RT2 Profiler PCR Arrays require Minimum 25 ng Total RNA

Positive Call Rate (CT < 35)
• 99% with 500 ng RNA
• 80% with 25 ng RNA

Human Inflammatory Cytokines & Receptors RT2 Profiler PCR Array

Small or Fixed Samples
A Challenge of RT-PCR

• Samples containing less than 25 ng
• Sources include:
• Stem Cells
• Fine Needle Biopsies/ Aspirations
• Laser Capture Microdissection
• Sorted Samples (FACS, selection markers, GFP)
• FFPE Samples
• Even though ample amounts of RNA can be
isolated, because of degradation and cross-linking
effects, only 1% of recovered RNA is considered
suitable for downstream gene expression analysis.


PCR Arrays with PreAMP – Increased Sensitivity
For Small & Fixed Samples

See More Genes with Even Less RNA – Pathway-Focused


How RT2 PCR Arrays Work
Small & FFPE Samples

Tandem PCR-based Technology
Small Samples (1-100ng RNA) / FFPE Samples (100 ng RNA)
gDNA Elimination Step
~45 min
First Strand Synthesis
(1/4 of RT product i.e. 5µL)
µ

1st strand cDNA

~40 min

+ PreAMP Primer Mix
and PreAMP Master Mix

PreAMP PCR Step
(Nano:12 Cycles
FFPE: 8 Cycles)

Amplified cDNA
~20 min

Total time:< 2 hr

2 hours

+ Side Reaction Reducer
(Exonuclease I)

Eliminate Residual
PreAMP Primers

Clean Amplified cDNA
+ SYBR Green Master Mix
Real-time PCR Detection of 89
Gene-specific Amplification on
RT2 Profiler PCR Array


Essentials of RT2 PreAMP Technology

~40 min

+ PreAMP Primer Mix
and PreAMP Master Mix

PreAMP PCR Step
(12/8 Cycles)

Amplified cDNA
~20 min

+ Side Reaction Reducer
(Exonuclease I)

Eliminate Residual
PreAMP Primers

96-well Pathway-Powered PCR Array
• 84 Genes of Interest
• 5 Housekeeping Genes
• 7 Controls
Each PreAMP Primer Mix
• 84 Genes of Interest
• 1 primer set / gene
• 84 primer sets combined

RT2 PreAMP: Pathway-Focused Gene Amplification
• Faithfully Amplify ONLY those Genes Appearing on Your Array

FFPE Samples
Gene Expression Challenges

RNA Quality
• Variable degree of RNA degradation in RNA preparation
• Sample handling
• Unbuffered fixative solutions
• Sub-Optimal Storage & Extraction methods
• Impurities Interfere with Enzymatic Activity
• Cross-linking with proteins resulting from chemical
modification by formaldehyde
• Target sequences not available for downstream processes
• Prevent enzymes from replicating RNA in a processive manner
• RNA is difficult to purify & performs poorly in enzymatic reactions

Reduced Effective qRT-PCR templates
High Volume (100s) of samples: Biomarkers/ Prognostic models

Minimal processing time is essential

RNeasy FFPE RNA Extraction Kit

Proteinase K Digestion: Without standard de-paraffinization, treatment with Proteinase
K in a unique buffer system at an elevated temperature reverses cross-links, breaks up
and dissolves the tissue sample, and releases the nucleic acids into solution.
Phase Extraction: 2 Steps
• QiaZol
• Guanidinium isothiocyanate
• Powerful protein denaturant: Inactivation of RNAses
• Acidic Phenol/Chloroform
• Partitioning of RNA into aqueous supernatant for separation (low pH)
At neutral pH, DNA, not RNA, partitions into the aqueous phase

FFPE Sample Gene Expression Analysis
How it Works


RT2 PreAMP
Increased Positive Call Rate

• Sample: Human Spleen RNA was reverse transcribed to cDNA with (red bars) or without PreAMPlification (purple bars) using
RT² PreAMP PCR Master Mix and RT² PreAMP Primer Mix.
• PCR Array: Human Cancer PathwayFinder
• Unamplified: 55% of genes were virtually undetectable; no genes in the 10-20 Ct range.
•FFPE PreAMPlified: Genes with Ct values >30 shift into the visible range of 10-30.


RT2 PreAMP
Detection of “Absent” Genes

• Sample: Human Spleen RNA was reverse transcribed to cDNA with (red bars) or without PreAMPlification (purple bars) using
RT² PreAMP PCR Master Mix and RT² PreAMP Primer Mix.
• PCR Array: Human Cancer PathwayFinder
• Unamplified: 14 genes classified as “Absent” (Ct>35)
• FFPE PreAMPlified: 14 genes NOW classified as “Present” (Ct<35)


RT2 PreAMP
Unbiased Amplification

• Sample: First-strand cDNA was synthesized from the extracted Human Spleen RNA samples, and 1/4 of the RT product was
used for pre-amplification with RT² PreAMP PCR Master Mix plus Human Cancer PathwayFinder RT² PreAMP Primer Mix.
Unamplified cDNA synthesized from the same spleen and intestine samples were used as the control. FFPE PreAMP amplified
and unamplified cDNA samples were then analyzed on the Human Cancer PathwayFinder PCR Array, with the threshold cycle
(Ct) obtained.
• PCR Array: Human Cancer PathwayFinder PCR Array
• Result: Highly Comparable Ct values between FFPE PreAMP and Non-PreAMP samples

What’s Next – After PCR Arrays
• Validate results with more sample and focused set of genes
– Custom PCR Arrays
• Identify miRNAs that are expressed or regulating your gene of interest
– miRNA PCR Arrays
• Assess Biological Impact
– Cignal™ Reporters
• Knockdown Analysis using RNAi
– QIAGEN Flexitube/Flexiplate siRNA and SureSilencing shRNA Plasmids
• Analyze interaction between DNA & nuclear proteins
– ChampionChIP Chromatin Immunoprecipitation PCR Arrays
• Quantify secreted proteins in blood plasma sera
– ELISArrays

Create Your Own PCR Arrays
Custom Formats
• Choose any gene from the Human, Mouse, Rat, Drosophila, Rhesus macaque, or Dog Genomes
96-well

384-well
•

Analyze the Genes Most Important To
Your Research

•

Available for any real-time PCR
instrument.

•

Wet-Bench Validated design &
performance.

•

2 to 3 week turn around time first
order

•

2 to 3 DAY turn around on re-order

•

12 Genes : 8 Samples

Same Data Analysis as Catalogued
Arrays




96 Genes : 1 Sample

Custom 100-discs also available

# of
samples x 4

Replicates
Technical & Biological

Technical
• Not necessary
Save Time & Money
• Reproducibility of the PCR Arrays is very high
• Results demonstrate that what you are seeing is a result
of biology, not technique.
• RTC & PPC show technical reproducibility on each plate,
and comparable across plates.

Biological
• Needed to verify the results are a result of biology
• At least 3 replicates per sample for statistical analysis
• p values


RT2 PCR Array System SUMMARY

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•

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Complete System
• RNA isolation kits, PCR Arrays, master mix, first strand kit, and free data analysis
software
• Systematic controls to monitor sample quality, RT and PCR efficiency
• 3 hours from RNA to data analysis
Breadth of Pathway Content
• Profile the genes and pathways that you really care about
• More than 100 pathways for human, mouse, and rat
• Customizable by gene, set, or pathway
Superb Real-Time PCR Performance
• Reproducibility, Sensitivity, Specificity, Reliability
Broad Application
• Cancer, Immunology, Neuroscience, Stem Cells & much more

Additional Resources

E-Learning Center:
Recorded Seminars

Ffpe pcr array

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