Sample to Insight
RT2 Profiler PCR Array Data Analysis Tutorial
Anisha KharkiaAssociate Product ManagerBiological Research Content Management - GeneGlobe
RT2 Profiler PCR Array Data Analysis Tutorial 1
Sample to Insight
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
Sample to Insight
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 Services or your local distributor.
Sample to Insight
RT2 Profiler PCR Array Data Analysis Tutorial Webinar;
Pathway-focused solutions for expression analysis
http://www.geneglobe.com http://www.qiagen.com/
Microbial DNA qPCR Arrays
4
Sample to Insight
GeneGlobe Data Analysis Center – new!
Helping you even more with your data analysis• Assign your samples into groups more easily
o Sample Managero Upload a Microsoft Excel file
• Display your results more professionallyo Improved plots and chartso No need to turn off pop-up blocker
• More easily design your next experimento 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
RT2 Profiler PCR Array Data Analysis Tutorial 5
Sample to Insight
PCR array anatomy
Plate formats – all instruments and content depth
4x96 1x384
Rotor-Gene Q
RT2 Profiler PCR Array Data Analysis Tutorial 6
Sample to Insight
PCR array anatomy
Pathway- and gene-specific assays and RT-PCR controls
RT2 Profiler PCR Array Data Analysis Tutorial 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-specific GOIs
Sample to Insight
8
Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
What we have covered5
Sample to Insight
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 calculationso Gene Y up- / downregulated by x-fold
RT2 Profiler PCR Array Data Analysis Tutorial 9
Sample to Insight
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 Viewo Set range starting at cycle two–three to one–two cycles before earliest amplificationo 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 selecting reference 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 Analysis Tutorial 10
Sample to Insight
Setting baseline
Linear view – select “Auto Calculated”
RT2 Profiler PCR Array Data Analysis Tutorial 11
Sample to Insight
Setting threshold
Log view – use the same threshold for all PCR arrays
RT2 Profiler PCR Array Data Analysis Tutorial 12
Sample to Insight
13
Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
What we have covered5
Sample to Insight
14
Organize raw Ct values
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 Analysis Tutorial
Sample to Insight
15
Organize raw Ct values
Custom RT2 Profiler PCR Arrays
RT2 Profiler PCR Array Data Analysis Tutorial
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
Sample to Insight
16
Organize raw Ct values
Individual RT2 qPCR Assays
RT2 Profiler PCR Array Data Analysis Tutorial
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
Sample to Insight
17
Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
What we have covered5
Sample to Insight
Sample experiment – experimental setupGroup three biological replicates into 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
RT2 Profiler PCR Array Data Analysis Tutorial 18
Sample to Insight
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
RT2 Profiler PCR Array Data Analysis Tutorial 19
Sample to Insight
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 + ∆Ct3
∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct
RT2 Profiler PCR Array Data Analysis Tutorial 20
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
Sample to Insight
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 Group3. 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 Analysis Tutorial 21
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
∆∆Ct = ∆CtGroup 2 – ∆Ctcontrol
∆∆Ct = ∆CtGroup 3 – ∆Ctcontrol
Sample to Insight
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 Group3. Calculate ∆∆CT for each gene between Groups4. Calculate Fold Change: 2-∆∆Ct
RT2 Profiler PCR Array Data Analysis Tutorial 22
∆∆Ct = ∆CtGroup 1 – ∆Ctcontrol
∆∆Ct = ∆CtGroup 2 – ∆Ctcontrol
∆∆Ct = ∆CtGroup 3 – ∆Ctcontrol
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
∆Ct + ∆Ct + ∆Ct3
Sample to Insight
23
Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
What we have covered5
Sample to Insight
GeneGlobe Data Analysis Center
RT2 Profiler PCR Array Data Analysis live demonstration
RT2 Profiler PCR Array Data Analysis Tutorial 24
https://www.qiagen.com/us/geneglobe
Sample to Insight
25
Uploading your data4
Agenda
Protocol, baseline and threshold overview1
Organizing your raw data2
Sample experiment3
What we have covered5
Sample to Insight
Summary
1. Set instrument in absolute quantification or standard curve mode ABI, Stratagene, Bio-Rad and Eppendorf instruments
2. Set baselineo Set automatically with adaptive baseline or set manually in linear view
3. Set thresholdo Lower two thirds of amplification plot in log view. Use same threshold for all PCR arrayso 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 datao 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 Analysis Tutorial 26
What have we covered today?
Sample to Insight
Summary
• New Sample Managero More easily assigns your samples into groups
• Improved plots and chartso Display your results more professionally
• Upgraded What’s Nexto Designs your next experiment
• Remember: some templates require assay catalog numbers
• Be sure to download and use the new Microsoft Excel templates
RT2 Profiler PCR Array Data Analysis Tutorial 27
New benefits
Sample to Insight
Thank You for Attending
.Questions? Comments? Suggestions?
.Please contact Technical Support
. US & Canada
. Email: [email protected]
GeneRead NGS System
New product for microbial research
RT2 Profiler PCR Array Data Analysis Tutorial 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 Services or your local distributor.
. Customers outside North America
. Email: [email protected]
. Webinar questions
. Email: [email protected]
Top Related