The document describes the process for analyzing data from RT2 Profiler PCR Array experiments, which includes: 1) defining the baseline and threshold for Ct value determination, 2) organizing the raw Ct values in a spreadsheet, 3) uploading the data to a free online analysis portal, 4) using the portal to calculate fold changes between groups and export the results. The analysis allows comparison of up to 100 experimental groups to a control and identifies differentially expressed genes.

1. RT2 Profiler™ PCR Array: Web-based
data analysis tutorial
Questions? Comments? or Suggestions?
Ask now or contact Technical Support
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M – F, 9 AM – 6 PM EST.
Telephone: 888-503-3187;
Email: support@SABiosciences.com
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International customer:
Email: SABIO@QIAGEN.COM
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Any webinar related questions: qiawebinars@qiagen.com
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Sample & Assay Technologies

2. Pathway-Focused Solution for Expression Analysis
http://sabiosciences.com/home.php
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Sample & Assay Technologies

3. Topics to be Covered
Topic I:
.
Brief Technology and Protocol Overview
Topic II (Today):
PCR Array Data Analysis
•
•
•
•
•
Define Baseline and Threshold
Web Portal Location / Address
Upload Raw Ct Data
Analyze Data & Controls
Export Results
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Sample & Assay Technologies

4. Anatomy of a PCR Array
4x96
370
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Sample & Assay Technologies

5. Anatomy of a PCR Array
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
.
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Sample & Assay Technologies

6. How RT2 Profiler PCR Arrays Work
Isolate Total RNA
cDNA Synthesis
.
Control
Hot Sauce
–
–
Genomic DNA Removal Step (5
min.)
Reverse Transcription Step (20
min.)
Load Plates
.
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
.
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
.
15 minutes from Raw Ct to
Fold Change Data
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Sample & Assay Technologies

7. How RT2 Profiler PCR Arrays Work
cDNA Synthesis
.
control
Hot Sauce
Genomic DNA Removal Step (5
min.)
Reverse Transcription Step (20
min.)
Load Plates
.
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
.
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
.
15 minutes from Raw Ct to
Fold Change Data
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Sample & Assay Technologies

8. How RT2 Profiler PCR Arrays Work
cDNA Synthesis
.
control
Hot Sauce
Genomic DNA Removal Step (5
min.)
Reverse Transcription Step (20
min.)
Load Plates
.
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
.
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
.
15 minutes from Raw Ct to
Fold Change Data
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Sample & Assay Technologies

9. How RT2 Profiler PCR Arrays Work
cDNA Synthesis
.
control
Hot Sauce
–
–
Genomic DNA Removal Step (5
min.)
Reverse Transcription Step (20
min.)
Load Plates
.
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
.
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
.
15 minutes from Raw Ct to
Fold Change Data
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Sample & Assay Technologies

10. How RT2 Profiler PCR Arrays Work
cDNA Synthesis
.
control
Hot Sauce
–
–
Genomic DNA Removal Step (5
min.)
Reverse Transcription Step (20
min.)
Load Plates
.
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
.
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
.
15 minutes from Raw Ct to
Fold Change Data
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Sample & Assay Technologies

11. How RT2 Profiler PCR Arrays Work
cDNA Synthesis
.
control
Hot Sauce
–
–
Genomic DNA Removal Step (5
min.)
Reverse Transcription Step (20
min.)
Load Plates
.
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
.
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
.
15 minutes from Raw Ct to
Fold Change Data
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Sample & Assay Technologies

12. How RT2 Profiler PCR Arrays Work
control
cDNA Synthesis
.
Hot Sauce
–
–
Genomic DNA Removal Step (5
min.)
Reverse Transcription Step (20
min.)
Load Plates
.
1 Sample per PCR Array
2 minutes with multi-channel pipet
Raw C(t) Values
.
Fold Change Results
.
Using ∆∆C(t) calculations
ABL1 is up/down regulated by x
fold
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Sample & Assay Technologies

13. Topics to be Covered
Topic I:
.
Brief Technology and Protocol Overview
Topic II (Today):
PCR Array Data Analysis
•
•
•
•
•
Define Baseline and Threshold
Web Portal Location / Address
Upload Raw Ct Data
Analyze Data & Controls
Export Results
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Sample & Assay Technologies

14. Define Baseline and Threshold
For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:
Baseline
• Use Automated Baseline
-(if your instrument has Adaptive Baseline function) OR
• Manually Set Baseline
-Using Linear View:
Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest
amplification (with highest cycle being cycle #15)
.
Threshold Value
• Use Log View
• Place in
1) Linear phase of amplification curve
2) Above background signal, but within lower half to one third of curve
.
Export Ct values to blank spread sheet (Excel).
.
Threshold Must Be Same Between Runs (important for PPC and RTC and
selecting house keeping genes)
)
.
*For Roche LC480: Use Second Derivative Maximum
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Sample & Assay Technologies

15. Define Baseline and Threshold
For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:
Baseline
• Use Automated Baseline
-(if your instrument has Adaptive Baseline function) OR
• Manually Set Baseline
-Using Linear View:
Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest
amplification (with highest cycle being cycle #15)
.
Threshold Value
• Use Log View
• Place in
1) Linear phase of amplification curve
2) Above background signal, but within lower half to one third of curve
.
Export Ct values to blank spread sheet (Excel).
.
Threshold Must Be Same Between Runs (important for PPC and RTC and
selecting house keeping genes)
)
.
*For Roche LC480: Use Second Derivative Maximum
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Sample & Assay Technologies

16. Define Baseline and Threshold
For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:
Baseline
• Use Automated Baseline
-(if your instrument has Adaptive Baseline function) OR
• Manually Set Baseline
-Using Linear View:
Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest
amplification (with highest cycle being cycle #15)
.
Threshold Value
• Use Log View
• Place in
1) Linear phase of amplification curve
2) Above background signal, but within lower half to one third of curve
.
Export Ct values to blank spread sheet (Excel).
.
Threshold Must Be Same Between Runs (important for PPC and RTC and
selecting house keeping genes)
.
*For Roche LC480: Use Second Derivative Maximum
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Sample & Assay Technologies

17. Define Baseline and Threshold
For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:
Baseline
• Use Automated Baseline
-(if your instrument has Adaptive Baseline function) OR
• Manually Set Baseline
-Using Linear View:
Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest
amplification (with highest cycle being cycle #15)
.
Threshold Value
• Use Log View
• Place in
1) Linear phase of amplification curve
2) Above background signal, but within lower half to one third of curve
.
Export Ct values to a blank spread sheet (Excel).
.
Threshold Must Be Same Between Runs (important for PPC and RTC and
selecting house keeping genes)
.
*For Roche LC480: Use Second Derivative Maximum
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Sample & Assay Technologies

18. Define Baseline and Threshold
For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*:
Baseline
• Use Automated Baseline
-(if your instrument has Adaptive Baseline function) OR
• Manually Set Baseline
-Using Linear View:
Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest
amplification (with highest cycle being cycle #15)
.
Threshold Value
• Use Log View
• Place in
1) Linear phase of amplification curve
2) Above background signal, but within lower half to one third of curve
.
Export Ct values to blank spread sheet (Excel), in a single column.
.
Threshold must be same between runs (Important for PPC, RTC
and selecting house keeping genes)
*For Roche LC480: Use Second Derivative Maximum
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Sample & Assay Technologies

19. Define Baseline and Threshold
*For Roche LC480: Use Second Derivative Maximum
Baseline
• Use Automated Baseline
-(if your instrument has Adaptive Baseline function) OR
• Manually Set Baseline
-Using Linear View:
Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest
amplification (with highest cycle being cycle #15)
.
Threshold Value
• Use Log View
• Place in
1) Linear phase of amplification curve
2) Above background signal, but within lower half to one third of curve
.
Export Ct values to blank spread sheet (Excel), in a single column.
.
Threshold Must Be Same Between Runs (important for PPC an
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Sample & Assay Technologies

20. Setting Baseline
Select “Auto Calculated”
Linear View
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Sample & Assay Technologies

21. Setting Threshold
Log View
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Sample & Assay Technologies

22. Setting Threshold
Use the same threshold for All PCR arrays
Threshold Line
C(t)
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Sample & Assay Technologies

23. 2 Ways to “CRUNCH” the Data
Excel Based Templates
•Free!
•Download from http://www.sabiosciences.com/pcrarraydataanalysis.php
•Good for 2 Group Comparisons (Control + Experimental)
•10 PCR Arrays
Web-Based Data Analysis
•Free!
•Upload Excel spreadsheet at:
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
•Good for 100 Group Comparisons (Control + 99 Experimental)
•100 PCR Arrays Total
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
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Sample & Assay Technologies

24. Web portal location
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
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Sample & Assay Technologies

25. Organize Raw C(t) values
Download Excel Template from SABiosciences’ Web Portal…or make your own.
Cataloged Array
Row 1
Sample Name
Column A:
Well Location
Column B,C,D,E…
Raw C(t) Values
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Sample & Assay Technologies

26. Organe Raw C(t) values
Download Excel Template from SABiosciences’ Web Portal…or make your own.
Custom Array
Row 1
Sample Name
Column A:
Well Location
Column B:
Gene Name
Column C,D,E…
Raw C(t) Values
Custom Array format can be adapted for Individual PCR Assays
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Sample & Assay Technologies

27. Organize Raw C(t) values
Download Excel Template from SABiosciences’ Web Portal…or make your own.
LEAVE BLANK
Individual Assays
Row 1
Sample Name
Column A:
Well Location
Column B:
Gene Name
Well #
Column C,D,E…
Raw C(t) Values
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Sample & Assay Technologies

28. Our Experiment – An Example
Control
A
B
A
Group 3
C
B
A
B
Group 2
C
B
Group 1
C
A
C
Time 0
3 biological replicates will be grouped into (3 groups + control)
(resting 6 hr)
Time 24 hours (resting)
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Sample & Assay Technologies

29. Our Experiment - Data Analysis Overview
Control
A
B
Group 1
C
A
B
Group 2
C
A
B
Group 3
C
A
B
C
3 biological replicates will be grouped into (3 groups + control)
1 PCR Array for Each Sample (12 PCR Arrays Total)
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Sample & Assay Technologies

30. Our Experiment - Data Analysis Overview
Control
Group 1
Group 2
B
A
C
A
B
C
A
B
A
C
A
B
C
A
B
B
Group 3
A
C
C
B
A
B
C
C
∆C(t)
C(t)GOI- C(t)HKG
1.
Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest)
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Sample & Assay Technologies

31. Our Experiment - Data Analysis Overview
Control
Group 1
Group 2
B
A
∆C(t)
C
A
B
C
A
B
A
C
A
B
C
A
∆C(t)
∆C(t)
∆C(t)+∆C(t)+∆C(t)
3
1.
2.
∆C(t)
∆C(t)
∆C(t)
∆C(t)
B
Group 3
B
∆C(t)
∆C(t)+∆C(t)+∆C(t)
3
∆C(t)+∆C(t)+∆C(t)
3
A
C
C
∆C(t)
B
A
B
∆C(t)
C
C
∆C(t) ∆C(t)
∆C(t)+∆C(t)+∆C(t)
3
Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest)
Calculate Average ∆ C(t) for each gene within a Group
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Sample & Assay Technologies

32. Our Experiment - Data Analysis Overview
Control
A
B
∆C(t)
Group 1
C
∆C(t)
∆C(t)
A
∆C(t)
B
∆C(t)
Group 2
A
C
∆C(t)
∆C(t)
B
Group 3
C
∆C(t)
∆C(t)
A
∆C(t)
B
C
∆C(t) ∆C(t)
∆C(t)+∆C(t)+∆C(t)
3
∆C(t)+∆C(t)+∆C(t)
3
∆C(t)+∆C(t)+∆C(t)
3
∆C(t)+∆C(t)+∆C(t)
3
1.
2.
3.
Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest)
Calculate Average ∆ C(t) for each gene within a Group
Calculate ∆∆ C(t) for each gene between Groups
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Sample & Assay Technologies

33. Our Experiment - Data Analysis Overview
Control
A
B
∆C(t)
Group 1
C
∆C(t)
∆C(t)
A
∆C(t)
B
∆C(t)
Group 2
A
C
∆C(t)
∆C(t)
B
Group 3
C
∆C(t)
∆C(t)
A
∆C(t)
B
C
∆C(t) ∆C(t)
∆C(t)+∆C(t)+∆C(t)
3
∆C(t)+∆C(t)+∆C(t)
3
∆C(t)+∆C(t)+∆C(t)
3
∆C(t)+∆C(t)+∆C(t)
3
1.
2.
3.
4.
Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest)
Calculate Average ∆ C(t) for each gene within a Group
Calculate ∆∆ C(t) for each gene between Groups
∆∆Ct)
∆∆
Calculate Fold Change: 2(-∆∆
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Sample & Assay Technologies

34. Our Experiment-Data Analysis Overview
Control
(resting 6 hr)
Time 24 hours (resting)
http://www.sabiosciences.com/manuals/PCRArrayWhitePaper_App.pdf
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Sample & Assay Technologies

35. Web portal support tools
Quick reference
1.Take a Test Run with a Sample Data Set
2. Play a movie
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
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Sample & Assay Technologies

36. Web portal support tools
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
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Sample & Assay Technologies

37. Upload Raw Ct Data
1.Choose experiment
2. Select Array by catalog number
3. Select excel file with raw CT data
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
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Sample & Assay Technologies

38. Analysis setup: define experimental design
Define array group
(control or group # or exclude)
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Sample & Assay Technologies

39. Analysis setup: PCR array reproducibility & sample quality
1. Select each group
2. Rotor Gene?
PreAMP?
3. Check PPC
4. Check RTC
5. Check GDC
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Sample & Assay Technologies

40. Analysis setup: PCR array reproducibility & sample quality
1. Select Each Group
2. Rotor Gene?
PreAMP?
3. Check PPC
Don’t be panic, contact us
4. Check RTC
5. Check GDC
- 40 -
Sample & Assay Technologies

41. Analysis setup: Select normalization gene
Select Normalization Method
- 41 -
Sample & Assay Technologies

42. Method 1: Classic normalization gene selection
Manual selection
- 42 -
Sample & Assay Technologies

43. How to select/ remove a normalization gene
Select normalization gene(s)
- 43 -
Sample & Assay Technologies

44. Method 2: automatic selection from the HKG Panel
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Sample & Assay Technologies

45. Method 2: Automatic selection from the HKG Panel
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Sample & Assay Technologies

46. Method 3: Automatic selection from whole plate
- 46 -
Sample & Assay Technologies

47. Data Setup: Data overview
- 47 -
Sample & Assay Technologies

48. Analysis: Analyze results
Average ∆Ct
Fold Change
- 48 -
Fold Regulation
Sample & Assay Technologies

49. Analysis: Analyze results
Comments
- 49 -
Sample & Assay Technologies

50. Visualization of Data
Please turn off your Pop-Up Blocker!!
- 50 -
Sample & Assay Technologies

51. Visualization of Data – Heat map
Choose Groups
Click update
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Sample & Assay Technologies

52. Visualization of Data – Scatter plot
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Sample & Assay Technologies

53. Visualization of Data – Volcano plot
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Sample & Assay Technologies

54. Visualization of Data - Clustergram
Clustergram
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Sample & Assay Technologies

55. Summary
1.Set machine in Absolute Quantification / or Standard Curve Mode
2.Set baseline: (ABI, Stratagene, Bio-Rad and Eppendorf*)
A. Adaptive (use auto)
3.Set threshold:
A. Lower 2/3rds of amplification plot in log view
B. Use same threshold for all PCR Arrays
*Roche LC480 use Second Derivative Maximum
4. Export data into excel spreadsheet. Paste raw C(t) values into correct form:
(sample row 1, C(t)s according to well location.)
5. Upload data to SABiosciences web portal, or download excel data analysis
spreadsheets
6. Analyze Data
A. Group Biological/Technical Replicates
B. QC criteria (PPC, RTC, GDC)
C. Focus on Stable Housekeeping Genes
D. Fold Change Data
E. Export Data and Publish results
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Sample & Assay Technologies

56. What’s Next
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Sample & Assay Technologies

57. Thank you for attending our webinar!
Questions? Comments? or Suggestions?
Please contact Technical Support
.
.
.
.
.
.
.
.
US & Canada
Telephone: 888-503-3187
Email: support@SABiosciences.com
International Customers
Telephone: 888-503-3187
Email: sabio@Qiagen.com
GeneRead NGS System
- 57 -
Sample & Assay Technologies