QIAGEN PCR Arrays Seminar

Sample & Assay Technologies

QIAGEN PCR Arrays

Pathway Focused Productsfor Research and Development

Andrew WongRegional Sales Application Scientist SEA

Sample & Assay Technologies- 2 -

QIAGEN Range of Pathway Focused Products

� RT2 Profiler PCR Arrays

Accurate and Sensitive Technology for Pathway-Focuses Gene Expression Analysis

� miScript miRNA PCR Arrays

Simultaneous Detection of Genome-Wide or Pathway-Focused miRNA in Post-Transcriptional Gene Regulation

� EpiTect DNA Methyl PCR Arrays

Simple, Fast and Reliable DNA Methylation Analysis without Bisulfite Conversion


What is Your Pathway?How is the gene expression of biological pathway be ing studied?


Analytical Power of 96/384-well PCR Array


> 150 Pathways Available


Supports Majority of Real-time PCR Instruments

� 96-Well Blocks: 7000, 7300, 7500, 7700� FAST 96-Well Blocks: 7500, 7900HT� FAST 384-Well Block: 7900HT� StepOnePlus and ViiA 7

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

� Mastercycler ep realplex 2/2S/4/4S

� Mx3000p, Mx3005p, Mx4000p

� Rotor-Gene Q

� BioMark

� LightCycler 480

� TP-800


What is Your Pathway?Pathway Focused Products for Research and Developem ent

Gene Expression Analysis

� RT2 Profiler PCR Array System

– Accurate and Sensitive Technology for Pathway Gene Expression Analysis


RT2 Profiler PCR Arrays

Pathway Focused Gene Expression Analysis

� The RT2 Profiler PCR Array System

� Quality Control & Wet Bench Validation

� Application of the RT2 Profiler PCR Arrays


The RT2 Profiler PCR Array System

RT2 Profiler PCR Array5 Component System

1.RNeasy Mini Kit

2.RT2 First Strand Kit

3.RT2 SYBR Green MasterMix

4.RT2 Profiler PCR Array

5.Data Analysis Program


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

96-well version (1 sample/PCR Array)384-well version (4 samples/PCR Array)

Anatomy of RT 2 Profiler PCR Array


Modification and Customization


Web-Based Software� No installation needed

From Raw Ct Values to Fold Change Results� Using ∆∆Ct Method

Multiple Analysis Formats� Scatter Plot� Volcano Plot� Clustergram� 3D Histogram

Also available as downloadable Excel templates

Data Analysis Software



Scatter Plot


Volcano Plot




Clustergram


All PCR Arrays are Wet-Bench Validated

Primer Assays in each PCR Array are tested for:

� High Specificity:

�Single Product Amplification �Constant Ct value for same amount of template

� High Sensitivity:

�As low as 25ng of Total RNA�1ng of Total RNA for PreAMP systems

� High Amplification Efficiency:

�Single Curve Analysis�Standard Curve Dilutions


High Specificity of Primer Assays


RT2 Profiler PCR Arrays require Minimum 25ng Total RNA

High Sensitivity of Primer Assays

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


Wide Dynamic Range of Detection


Conclusion: Accurate and reliable ∆∆Ct results are guaranteed.

Representative set of 500 out of > 4,000 assays used in PCR Arrays

High PCR Amplification Efficiency

CycleGene 1

(AE 100%)Gene 2

(AE 85%)

0 1 1

1 2 1.85

2 4 3.42

..

35 3.4 x 1010 2.2 x 109

∆ 15x Difference

Why this matters?


User and Lot Reproducibility

Human Drug Metabolism PCR Array with Universal Reference RNA

Reproducibility Among Different Users

Minimal Lot-to-Lot Variability of PCR Arrays Manufa ctured on Different Days

� PCR Arrays manufactured using precision robots on two different days

� 2 separate users (4 Arrays each)

� All run on same machine, different days

� Baseline and threshold set the same across all PCR Arrays


Reproducibility Among Different Instruments

Minimal Instrument Variation

Human Drug Metabolism PCR Array with Universal Reference RNA

Minimal Instrument-to-Instrument Variability for PC R Arrays

� 2 different RNA samples

� 3 different PCR Instruments

� 2 different PCR Array Plate Types� (ABI 7500 vs Mx3000 and iCycler)

� 2 Different Mastermixes � (ABI7500 and Mx3000 vs iCycler)

� Standard cycling conditions on ALL machines

� Same Fold Regulation Results on All machines


Application PCR Arrays in Cancer Treatment Studies

rhTRAIL

Resistant to Apoptosis(Some Cells)

Apoptosis(Most Cells)

Cancer Cells

Question: What changes in gene expression occurred in resistant cells that can explain the mechanism of resistant to apoptosis?



� Highly sensitive to TRAIL induced apoptosis at optimal TRAIL concentration� Low concentration of TRAIL results in TRAL-induced apoptosis resistant cells

What Are the gene expression changes that occur during exposure to Low Doses of rhTRAIL?

How are the changes to gene expression that is driving the resistance to apoptosis?

Use SABiosciences' Apoptosis 384-well HT qPCR Arrays

MDA-MB-231 cells

Resistant to rhTRAIL(Survived)

High dose

Low dose

rhTRAIL

Apoptosis

MDA-MB-231 Cells



MDA-MB-231 cells

Resistant to rhTRAIL(Survived)

High dose

Low dose

rhTRAIL

Apoptosis

Results: qPCR reveals up regulation of c-FLIP, Stat5a and Stat5b, Bcl-XL and cyclin D1.

� c-Flip antagonizes caspase 8 activation

� Stat5b responsible for BCL-XL and Cyclin D1 transcr iption

� BCL-XL is anti-apoptosis protein


Stress & Toxicity PathwayFinder™ PCR Array

Uncovered Distinct Gene Expression Profiles Associated with Liver Toxicity Caused by 3 PPARγ Agonists (Actos, Avandia, and Rezulin)

Application PCR Arrays in Toxicity Studies



microRNA Analysis

� miScript miRNA PCR Array System

– Simultaneous Detection of Genome-Wide or Pathway-Focused miRNA in Post-Transcriptional Gene Regulation


miR-

142-5p

miR-

16

miR-

142-3p

miR-

21

miR-

15a

miR-

29b

Let-

7a

miR-

126

miR-

143

miR-

27a

Let-

7b

Let-

7f

miR-

9

miR-

26a

miR-

24

miR-

30e

miR-

181a

miR-

29a

miR-

124

miR-

144

miR-

30d

miR-

19b

miR-

22

miR-

122

miR-

150

miR-

32

miR-

155

miR-

140-5p

miR-

125b

miR-

141

miR-

92a

miR-

424

miR-

191

miR-

17

miR-

130a

miR-

20a

miR-

27b

miR-

26b

miR-

146a

miR-

200c

miR-

99a

miR-

19a

miR-

23a

miR-

30a

Let-

7i

miR-

93

miR-

106b

Let-

7c

miR-

101

Let-

7g

miR-

425

miR-

15b

miR-

28-5p

miR-

18a

miR-

25

miR-

23b

miR-

302a

miR-

186

miR-

29c

miR-

7

Let-

7d

miR-

30c

miR-

181b

miR-

223

miR-

320

miR-

374a

miR-

151-5p

Let-

7e

miR-

374b

miR-

196b

miR-

140-3p

miR-

100

miR-

103

miR-

96

miR-

302b

miR-

194

miR-

125a-5p

miR-

423-5p

miR-

376c

miR-

195

miR-

222

miR-

28-3p

miR-

128a

miR-

302c

PPCMi-

RTCPPC

Mi-

RTCU6

SNORD

44

SNORD

47

SNORD

48

miR-

423-3p

miR-

185

miR-

30b

miR-

210

Anatomy of a miRNA PCR Array

88 Pathway-Specific miRNAs

4 “Housekeeping” miRNAs

miRNA Reverse Transcription Controls (miRTC) n=2

Positive PCR Controls (PPC) n=2

Human miFinder miRNA PCR Array (MAH-001)


How RT2 miRNA PCR Arrays Work

cDNA Synthesis from miRNA� 2 hours

Load Plates(Preferably with 8-Channel Pipettors)

� 2 minutes

Run 40 cycle qPCR Program � 2 hours

Upload and Analyze Data� 15 minutes


QIAGEN Universal miRNA Reverse Transcription

miRNA Specific Forward Primer

Universal Reverse Primer


Advantages of Universal Reverse Transcription

� Ease of Screening

� Simpler setup

� Less sample necessary for analyses

� Equal RT reaction for each miRNA

� Comprehensive miRNA coverage

� cDNA can be saved for new analyses when additional miRNA sequences are discovered in the future

� 3’ Dicer processing is heterogeneous, causing multiple miRNA variants of each species, identifiable with universal RT

Similar potential specificity, & additional flexibility!



Epigenetic Analysis

� EpiTect DNA Methyl PCR Array System

– Simple, Fast and Reliable DNA Methylation Analysis without Bisulfite Conversion


Blood, 93:4059-4070, 1999

DNA Methylation and Transcriptional Repression


Challenges in Existing Technologies

� Bisulfite Treatment

� Converts all unmethylated cytosines into uracil

� Methylated cytocines remain unaffected

� Uracil is replaced by thymine during PCR reaction

� Downside to Bisulfite Treatment

� DNA fragmentation– Low PCR sensitivity– High PCR failure

� Incomplete bisulfite conversion– False positive methylation results

� Time consuming, laborious, low throughput and high expenses.


Methyl-Profiler PCR System – The Principle

Ordway et al., (2006) Carcinogenesis 27(12): 2409


(0.5 – 4.0 ug of Genomic DNA)

Note: The same DNA sample and same primer set for a targeted region of interest is used in each of the enzyme reactions.

e.g. A1/C1/E1/G1 amplify the same target from each of the four enzyme reactions.

Methyl-Profiler PCR System – The Protocol


QIAGEN Range of Pathway Focused Products


SABiosciences Online Resources


SABiosciences Online Webinars


SABiosciences Pre -Recorded Webinars


Peer-Reviewed Publications


Gene Network Central Pro


[email protected]

QIAGEN PCR Arrays Seminar

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