Download - QIAGEN PCR Arrays Seminar
Sample & Assay Technologies
QIAGEN PCR Arrays
Pathway Focused Productsfor Research and Development
Andrew WongRegional Sales Application Scientist SEA
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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
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What is Your Pathway?How is the gene expression of biological pathway be ing studied?
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Analytical Power of 96/384-well PCR Array
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> 150 Pathways Available
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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
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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
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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
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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
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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
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Modification and Customization
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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
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Data Analysis Software
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Data Analysis Software
Scatter Plot
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Volcano Plot
Data Analysis Software
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Data Analysis Software
Clustergram
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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
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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
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High Specificity of Primer Assays
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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
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Wide Dynamic Range of Detection
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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?
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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
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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
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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
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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?
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Application PCR Arrays in Cancer Treatment Studies
� 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
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Application PCR Arrays in Cancer Treatment Studies
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
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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
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What is Your Pathway?Pathway Focused Products for Research and Developem ent
microRNA Analysis
� miScript miRNA PCR Array System
– Simultaneous Detection of Genome-Wide or Pathway-Focused miRNA in Post-Transcriptional Gene Regulation
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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)
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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
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QIAGEN Universal miRNA Reverse Transcription
miRNA Specific Forward Primer
Universal Reverse Primer
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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!
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What is Your Pathway?Pathway Focused Products for Research and Developem ent
Epigenetic Analysis
� EpiTect DNA Methyl PCR Array System
– Simple, Fast and Reliable DNA Methylation Analysis without Bisulfite Conversion
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Blood, 93:4059-4070, 1999
DNA Methylation and Transcriptional Repression
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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.
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Methyl-Profiler PCR System – The Principle
Ordway et al., (2006) Carcinogenesis 27(12): 2409
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(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
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QIAGEN Range of Pathway Focused Products
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SABiosciences Online Resources
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SABiosciences Online Webinars
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SABiosciences Pre -Recorded Webinars
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Peer-Reviewed Publications
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Peer-Reviewed Publications
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Gene Network Central Pro
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Gene Network Central Pro
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Gene Network Central Pro
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