- 1.RT2 Profiler PCR Array: Web-based data analysis
tutorialQuestions? Comments? or Suggestions? Ask now or contact
Technical Support..M F, 9 AM 6 PM EST. Telephone: 888-503-3187;
Email: [email protected] customer: Email:
[email protected] webinar related questions:
[email protected] & Assay Technologies
2. Pathway-Focused Solution for Expression
Analysishttp://sabiosciences.com/home.php -2-Sample & Assay
Technologies 3. Topics to be Covered Topic I:.Brief Technology and
Protocol OverviewTopic II (Today): PCR Array Data Analysis Define
Baseline and Threshold Web Portal Location / Address Upload Raw Ct
Data Analyze Data & Controls Export Results-3-Sample &
Assay Technologies 4. Anatomy of a PCR Array4x96370-4-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 .-5-Sample & Assay Technologies 6. How RT2
Profiler PCR Arrays Work Isolate Total RNA cDNA
Synthesis.ControlHot Sauce Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)Load Plates.1 Sample per PCR
Array 2 minutes with multi-channel pipetRun 40 cycle qPCR
Program.Standard cycling conditions for all Real Time PCR
Instruments 2 hoursUpload and Analyze Data (FREE).15 minutes from
Raw Ct to Fold Change Data-6-Sample & Assay Technologies 7. How
RT2 Profiler PCR Arrays Work cDNA Synthesis.controlHot SauceGenomic
DNA Removal Step (5 min.) Reverse Transcription Step (20 min.)Load
Plates.1 Sample per PCR Array 2 minutes with multi-channel pipetRun
40 cycle qPCR Program.Standard cycling conditions for all Real Time
PCR Instruments 2 hoursUpload and Analyze Data (FREE).15 minutes
from Raw Ct to Fold Change Data-7-Sample & Assay Technologies
8. How RT2 Profiler PCR Arrays Work cDNA Synthesis.controlHot
SauceGenomic DNA Removal Step (5 min.) Reverse Transcription Step
(20 min.)Load Plates.1 Sample per PCR Array 2 minutes with
multi-channel pipetRun 40 cycle qPCR Program.Standard cycling
conditions for all Real Time PCR Instruments 2 hoursUpload and
Analyze Data (FREE).15 minutes from Raw Ct to Fold Change
Data-8-Sample & Assay Technologies 9. How RT2 Profiler PCR
Arrays Work cDNA Synthesis.controlHot Sauce Genomic DNA Removal
Step (5 min.) Reverse Transcription Step (20 min.)Load Plates.1
Sample per PCR Array 2 minutes with multi-channel pipetRun 40 cycle
qPCR Program.Standard cycling conditions for all Real Time PCR
Instruments 2 hoursUpload and Analyze Data (FREE).15 minutes from
Raw Ct to Fold Change Data-9-Sample & Assay Technologies 10.
How RT2 Profiler PCR Arrays Work cDNA Synthesis.controlHot Sauce
Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20
min.)Load Plates.1 Sample per PCR Array 2 minutes with
multi-channel pipetRun 40 cycle qPCR Program.Standard cycling
conditions for all Real Time PCR Instruments 2 hoursUpload and
Analyze Data (FREE).15 minutes from Raw Ct to Fold Change Data- 10
-Sample & Assay Technologies 11. How RT2 Profiler PCR Arrays
Work cDNA Synthesis.controlHot Sauce Genomic DNA Removal Step (5
min.) Reverse Transcription Step (20 min.)Load Plates.1 Sample per
PCR Array 2 minutes with multi-channel pipetRun 40 cycle qPCR
Program.Standard cycling conditions for all Real Time PCR
Instruments 2 hoursUpload and Analyze Data (FREE).15 minutes from
Raw Ct to Fold Change Data- 11 -Sample & Assay Technologies 12.
How RT2 Profiler PCR Arrays Work controlcDNA 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 pipetRaw C(t) Values.Fold Change Results.Using C(t)
calculations ABL1 is up/down regulated by x fold- 12 -Sample &
Assay Technologies 13. Topics to be Covered Topic I:.Brief
Technology and Protocol OverviewTopic II (Today): PCR Array Data
Analysis Define Baseline and Threshold Web Portal Location /
Address Upload Raw Ct Data Analyze Data & Controls Export
Results- 13 -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 - 14 -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 - 15 -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 - 16 -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 - 17 -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 - 18 -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 - 19 -Sample & Assay
Technologies 20. Setting Baseline Select Auto CalculatedLinear
View- 20 -Sample & Assay Technologies 21. Setting ThresholdLog
View- 21 -Sample & Assay Technologies 22. Setting ThresholdUse
the same threshold for All PCR arraysThreshold LineC(t)- 22 -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 ArraysWeb-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
Totalhttp://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
- 23 -Sample & Assay Technologies 24. Web portal
locationhttp://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
- 24 -Sample & Assay Technologies 25. Organize Raw C(t) values
Download Excel Template from SABiosciences Web Portalor make your
own.Cataloged Array Row 1 Sample NameColumn A: Well LocationColumn
B,C,D,E Raw C(t) Values- 25 -Sample & Assay Technologies 26.
Organe Raw C(t) values Download Excel Template from SABiosciences
Web Portalor 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 - 26
-Sample & Assay Technologies 27. Organize Raw C(t) values
Download Excel Template from SABiosciences Web Portalor make your
own. LEAVE BLANKIndividual Assays Row 1 Sample Name Column A: Well
Location Column B: Gene NameWell #Column C,D,E Raw C(t) Values- 27
-Sample & Assay Technologies 28. Our Experiment An Example
Control A BAGroup 3CB ABGroup 2 CBGroup 1CA CTime 0 3 biological
replicates will be grouped into (3 groups + control) (resting 6 hr)
Time 24 hours (resting) - 28 -Sample & Assay Technologies 29.
Our Experiment - Data Analysis Overview Control ABGroup 1 CABGroup
2 CABGroup 3 CABC3 biological replicates will be grouped into (3
groups + control)1 PCR Array for Each Sample (12 PCR Arrays Total)-
29 -Sample & Assay Technologies 30. Our Experiment - Data
Analysis Overview ControlGroup 1Group 2BACABCABACABCAB BGroup 3 AC
CBABC CC(t) C(t)GOI- C(t)HKG1.Calculate C(t) for on each array for
each GOI (Gene Of Interest)- 30 -Sample & Assay Technologies
31. Our Experiment - Data Analysis Overview ControlGroup 1Group 2BA
C(t)CABCABACABCAC(t)C(t)C(t)+C(t)+C(t) 31. 2.C(t)C(t)C(t)C(t)BGroup
3B C(t)C(t)+C(t)+C(t) 3C(t)+C(t)+C(t) 3AC C C(t)BABC(t)C CC(t)
C(t)C(t)+C(t)+C(t) 3Calculate C(t) for on each array for each GOI
(Gene Of Interest) Calculate Average C(t) for each gene within a
Group- 31 -Sample & Assay Technologies 32. Our Experiment -
Data Analysis Overview Control ABC(t)Group 1 CC(t)C(t)A C(t)B
C(t)Group 2 AC C(t)C(t)BGroup 3 CC(t)C(t)A C(t)BC C(t)
C(t)C(t)+C(t)+C(t) 3 C(t)+C(t)+C(t) 3C(t)+C(t)+C(t) 3C(t)+C(t)+C(t)
31. 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- 32 -Sample & Assay
Technologies 33. Our Experiment - Data Analysis Overview Control
ABC(t)Group 1 CC(t)C(t)A C(t)B C(t)Group 2 AC C(t)C(t)BGroup 3
CC(t)C(t)A C(t)BC C(t) C(t)C(t)+C(t)+C(t) 3 C(t)+C(t)+C(t)
3C(t)+C(t)+C(t) 3C(t)+C(t)+C(t) 31. 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(- - 33 -Sample & Assay
Technologies 34. Our Experiment-Data Analysis OverviewControl
(resting 6 hr) Time 24 hours (resting)
http://www.sabiosciences.com/manuals/PCRArrayWhitePaper_App.pdf -
34 -Sample & Assay Technologies 35. Web portal support
toolsQuick reference1.Take a Test Run with a Sample Data Set2. Play
a
moviehttp://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
- 35 -Sample & Assay Technologies 36. Web portal support
toolshttp://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
- 36 -Sample & Assay Technologies 37. Upload Raw Ct
Data1.Choose experiment2. Select Array by catalog number3. Select
excel file with raw CT
datahttp://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php
- 37 -Sample & Assay Technologies 38. Analysis setup: define
experimental designDefine array group (control or group # or
exclude)- 38 -Sample & Assay Technologies 39. Analysis setup:
PCR array reproducibility & sample quality1. Select each group
2. Rotor Gene? PreAMP? 3. Check PPC 4. Check RTC 5. Check GDC- 39
-Sample & Assay Technologies 40. Analysis setup: PCR array
reproducibility & sample quality1. Select Each Group 2. Rotor
Gene? PreAMP? 3. Check PPC Dont be panic, contact us4. Check RTC 5.
Check GDC- 40 -Sample & Assay Technologies 41. Analysis setup:
Select normalization geneSelect Normalization Method- 41 -Sample
& Assay Technologies 42. Method 1: Classic normalization gene
selectionManual selection- 42 -Sample & Assay Technologies 43.
How to select/ remove a normalization geneSelect normalization
gene(s)- 43 -Sample & Assay Technologies 44. Method 2:
automatic selection from the HKG Panel- 44 -Sample & Assay
Technologies 45. Method 2: Automatic selection from the HKG Panel-
45 -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 CtFold Change- 48 -Fold
RegulationSample & Assay Technologies 49. Analysis: Analyze
results Comments- 49 -Sample & Assay Technologies 50.
Visualization of DataPlease turn off your Pop-Up Blocker!!- 50
-Sample & Assay Technologies 51. Visualization of Data Heat
mapChoose GroupsClick update- 51 -Sample & Assay Technologies
52. Visualization of Data Scatter plot- 52 -Sample & Assay
Technologies 53. Visualization of Data Volcano plot- 53 -Sample
& Assay Technologies 54. Visualization of Data - Clustergram
Clustergram- 54 -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- 55 -Sample & Assay Technologies 56. Whats Next- 56
-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: [email protected] International Customers Telephone:
888-503-3187 Email: [email protected] NGS System- 57 -Sample
& Assay Technologies
