This webinar presentation provides an overview and tutorial on analyzing data from RT2 Profiler PCR Array experiments. It discusses organizing raw Ct value data, performing ΔCt and ΔΔCt calculations to analyze gene expression changes between sample groups, and using the GeneGlobe Data Analysis Center web portal to analyze the data. The webinar highlights new features of the Data Analysis Center including improved data visualization and an upgraded sample manager. It emphasizes following the standard protocol for setting baselines and thresholds when analyzing PCR array data.

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RT2 Profiler PCR Array Data Analysis Tutorial
Anisha Kharkia
Associate Product Manager
Biological Research Content Management- GeneGlobe
RT2 Profiler PCR Array Data AnalysisTutorial 1

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Welcome to our four-part webinar series on qPCR
2
qPCR technology overview, applications, data
analysis and service solutions
• Part 1: Introduction to Real-Time PCR (Q-PCR / qPCR/ qrt-PCR)
• Part 2: Advanced Real-Time PCR Array Technology – Coding and Noncoding
RNA Expression Analysis
• Part 3: PCR Array Data Analysis Tutorial
• Part 4: Accelerate Your Discovery With QIAGEN Service Solutions for Biomarker
Research

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Legal disclaimer
QIAGEN products shown here are intended for molecular biology applications. These products are not
intended for the diagnosis, prevention or treatment of a disease.
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit
handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or
can be requested from QIAGEN Technical Servicesor your local distributor.

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Pathway-focused solutions for expression analysis
http://www.geneglobe.com http://www.qiagen.com/
Microbial DNA qPCR Arrays
RT2 Profiler PCR Array Data AnalysisTutorial Webinar; 4

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GeneGlobe Data Analysis Center – new!
Helping you even more with your data analysis
• Assign your samples into groups more easily
o Sample Manager
o Upload a Microsoft Excel file
• Display your results more professionally
o Improved plots and charts
o No need to turn off pop-up blocker
• More easily design your next experiment
o Upgraded What’s Next
• Applied to all array and assay technologies
What else you need to know
• Upgraded custom PCR arrays and individual qPCR assays
• Data upload templates require assay catalog numbers
o Be sure to download and use the new Microsoft Excel templates
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PCR array anatomy
Plate formats – all instruments and contentdepth
4x96
1x384
Rotor-Gene Q
RT2 Profiler PCR Array Data AnalysisTutorial 6

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PCR array anatomy
Pathway- and gene-specific assays and RT-PCR controls
RT2 Profiler PCR Array Data AnalysisTutorial 7
• Pathway-specific genes of
interest (GOIs) (84)
• Reference (HKG) gene controls
(5)
• Genomic DNA control (GDC)
• Reverse transcription controls
(RTCs) (3)
• Positive PCR controls (PPCs) (3)
Choose your reference genes
• Manually
• Automatically from reference gene panel or pathway-specificGOIs

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Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
8
What we have covered5

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How RT2 Profiler PCR Arrays work
Brief Protocol Overview
Control Sample
Synthesize cDNA
• Genomic DNA removal step (5 min)
• Reverse transcription step (20 min)
Load plates
• One sample per PCR array
• Two minutes with multi-channel pipet
Run 40-cycle qPCR program
• Standard cycling conditions
• All real-time PCR instruments
• Two hours
Upload and analyze data (FREE)
• Raw Ct Values
• Fold Change Results
o Using ∆∆Ct calculations
o Gene Y up- / downregulatedby x-fold
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Define baseline and threshold
Baseline(for ABI, Stratagene, Bio-Rad and Eppendorf instruments)
• Use automated baseline if your instrument has adaptive baseline function or
• Manually Set Baseline
o Using Linear View
o Set range starting at cycle two–three to one–two cycles before earliest amplification
o No higher than cycle 15
Threshold value
• Use log view
• Place in linear phase of amplification curve
o Above background signal within lower half to one third of curve
• Threshold must be same between runs
o Important for PPC and RTC interpretation and for selectingreference genes
Roche LC480 instruments
• Use second derivative maximum
• Export Ct values to a blank spreadsheet (Excel), in a single column per array
RT2 Profiler PCR Array Data AnalysisTutorial 10

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Setting baseline
Linear view – select“Auto Calculated”
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Setting threshold
Log view – use the same threshold for all PCR arrays
RT2 Profiler PCR Array Data AnalysisTutorial 12

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Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
13
What we have covered5

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Organize raw Ct values
14
Cataloged PCR arrays
Row one
• Sample name
Row two
• Groupings
Column A
• Well location
Column B, C, D, etc.
• Raw Ct values for each
sample
RT2 Profiler PCR Array Data AnalysisTutorial

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Organize raw Ct values
15
Custom RT2 Profiler PCR Arrays
RT2 Profiler PCR Array Data AnalysisTutorial
Row one
• Sample Name
Row two
• Groupings
Column A
• Well Location
Column B
• Gene Symbols
Column C
• Assay catalog numbers
Column D, E, F, etc.
• Raw Ct values
for each sample

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Organize raw Ct values
16
Individual RT2 qPCR Assays
RT2 Profiler PCR Array Data AnalysisTutorial
Row one
• Sample Name
Row two
• Groupings
Column A
• Assay numbering
Column B
• Gene symbols
Column C
• Assay catalog numbers
Column D, E, F, etc.
• Raw Ct values
for each sample

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Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
17
What we have covered5

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Sample experiment – experimental setup
Group three biological replicatesinto three groups and one control
A B C Control: resting 6 h
A B C Group 1: PMA + Ionomycin 6 h
A B C Group 2: resting 24 h
CA B Group 3: PMA + Ionomycin 24 h
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Sample experiment – data analysis overview
A
Group 3
B CA
Group 2
B CA
Group 1
B CA
Control
CB
CtGOI – CtHKG
∆Ct
1. Calculate ∆Ct on each array for each GOI (Gene Of Interest)
A B C A B C A B C A B C
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Sample experiment – data analysis overview
A
Group 3
B CA
Group 2
B CA
Group 1
B CA
Control
CB
A B C A B C A B C A B C
1. Calculate ∆Ct on each array for each GOI (Gene Of Interest)
2. Calculate average ∆Ct for each gene within a group
∆Ct + ∆Ct + ∆Ct
3
∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct
RT2 Profiler PCR Array Data AnalysisTutorial 20
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3

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Sample experiment – data analysis overview
A B C A B C A B C A B C
1. Calculate ∆Ct on each array for each GOI (Gene Of Interest)
2. Calculate Average ∆Ct for each gene within a Group
3. Calculate ∆∆Ct for each gene between Groups
∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct
∆∆Ct = ∆CtGroup 1 – ∆Ctcontrol
RT2 Profiler PCR Array Data AnalysisTutorial 21
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3
∆∆Ct = ∆CtGroup 2 – ∆Ctcontrol
∆∆Ct = ∆CtGroup 3 – ∆Ctcontrol

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Sample experiment – data analysis overview
A B C A B C A B C A B C
∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct
1. Calculate ∆CT on each array for each GOI (Gene Of Interest)
2. Calculate Average ∆CT for each gene within a Group
3. Calculate ∆∆CT for each gene between Groups
4. Calculate Fold Change: 2-∆∆Ct
RT2 Profiler PCR Array Data AnalysisTutorial 22
∆∆Ct = ∆CtGroup 1 – ∆Ctcontrol
∆∆Ct = ∆CtGroup 2 – ∆Ctcontrol
∆∆Ct = ∆CtGroup 3 – ∆Ctcontrol
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3
∆Ct + ∆Ct + ∆Ct
3

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Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
23
What we have covered5

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GeneGlobe Data Analysis Center
RT2 Profiler PCRArrayData Analysis live demonstration
RT2 Profiler PCR Array Data AnalysisTutorial 24
https://www.qiagen.com/us/geneglobe

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Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
25
What we have covered5

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Summary
1. Set instrument in absolute quantification or standard curve mode
✓ ABI, Stratagene, Bio-Rad and Eppendorf instruments
2. Set baseline
o Set automatically with adaptive baseline or set manually in linear view
3. Set threshold
o Lower two thirds of amplification plot in log view. Use same threshold for all PCR arrays
o Roche LC480: for items two and three use second derivative maximum
4. Export data into Microsoft Excel. Paste raw Ct values into correct format
5. Upload data to GeneGlobe Data Analysis Center web portal
6. Analyze data
o Group technical replicates of same biological condition together
o Check QC criteria (PPC, RTC, GDC)
o Identify stable reference genes
o Review fold change data
o Export data and publish results
RT2 Profiler PCR Array Data AnalysisTutorial 26
What have we covered today?

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Summary
• New Sample Manager
o More easily assigns your samples into groups
• Improved plots and charts
o Display your results more professionally
• Upgraded What’s Next
o Designs your next experiment
• Remember: some templates require assaycatalog numbers
• Be sure to download and use the new Microsoft Excel templates
RT2 Profiler PCR Array Data AnalysisTutorial 27
New benefits

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Thank You for Attending
.Questions? Comments? Suggestions?
.Please contact Technical Support
. US & Canada
. Email: BRCsupport@qiagen.com
GeneRead NGS System
New productfor microbial research
RT2 Profiler PCR Array Data AnalysisTutorial 28
For up-to-date licensing information and product-
specific disclaimers, see the respective QIAGEN kit
handbook or user manual. QIAGEN kit handbooks and
user manuals are available at www.QIAGEN.com or
can be requested from QIAGEN Technical Servicesor
your local distributor.
.Customers outside North America
.Email: SABIO@qiagen.com
.Webinar questions
.Email: QIAWebinars@qiagen.com