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RT2 Profiler PCR Array Data Analysis Tutorial

Anisha KharkiaAssociate Product ManagerBiological Research Content Management - GeneGlobe

RT2 Profiler PCR Array Data Analysis Tutorial 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 Services or your local distributor.

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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

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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 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

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PCR array anatomy

Plate formats – all instruments and content depth

4x96 1x384

Rotor-Gene Q

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PCR array anatomy

Pathway- and gene-specific assays and RT-PCR controls

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• 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

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Uploading your data4

Agenda

Protocol, baseline and threshold overview1

Organizing your raw data2

Sample experiment3

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 calculationso Gene Y up- / downregulated by 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 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

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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

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Uploading your data4

Agenda

Protocol, baseline and threshold overview1

Organizing your raw data2

Sample experiment3

What we have covered5

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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

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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

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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

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Uploading your data4

Agenda

Protocol, baseline and threshold overview1

Organizing your raw data2

Sample experiment3

What we have covered5

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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

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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 + ∆Ct3

∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct

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∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

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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 Group3. Calculate ∆∆Ct for each gene between Groups

∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct

∆∆Ct = ∆CtGroup 1 – ∆Ctcontrol

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∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

∆∆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 Group3. Calculate ∆∆CT for each gene between Groups4. Calculate Fold Change: 2-∆∆Ct

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∆∆Ct = ∆CtGroup 1 – ∆Ctcontrol

∆∆Ct = ∆CtGroup 2 – ∆Ctcontrol

∆∆Ct = ∆CtGroup 3 – ∆Ctcontrol

∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

∆Ct + ∆Ct + ∆Ct3

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Uploading your data4

Agenda

Protocol, baseline and threshold overview1

Organizing your raw data2

Sample experiment3

What we have covered5

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GeneGlobe Data Analysis Center

RT2 Profiler PCR Array Data Analysis live demonstration

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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

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 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

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What have we covered today?

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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

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New benefits

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Thank You for Attending

.Questions? Comments? Suggestions?

.Please contact Technical Support

. US & Canada

. Email: BRCsupport@qiagen.com

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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: SABIO@qiagen.com

. Webinar questions

. Email: QIAWebinars@qiagen.com