genex qpcr data analysis 2009-03-10 - gene-quantification · genex standard includes modules for:...
TRANSCRIPT
![Page 1: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/1.jpg)
GenEx qPCR Data Analysis
2009-03-10
![Page 2: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/2.jpg)
Presentation Outline
� qPCR Data Analysis
• Absolute Quantification
• Relative Quantification
• Expression Profiling
� GenEx Software Tool� GenEx Software Tool
• Modules
• Pre-Processing
• Statistical Tests
• Visualizations
![Page 3: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/3.jpg)
qPCR Data Analysis
5
10
15
20
25
30
0 1 2 3 4 5 6 7 8
?
Absolute Quantification
Relative Quantification
Expression Profiling
?
A B
?
?
?
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GenEx Standard includes modules for:
� Data editor• Data corrections
• Normalizations
• Scaling
� Data manager• Definitions of groups and categories
• Control of presentation parameters
• Scaling
� Plot functions• 2D/3D functionalities• 2D/3D functionalities
• Line plots, Box plots, scatterplots and more
� Reference gene identification• geNorm
• NormFinder
� Statistics• Descriptive statistics
• Student’s t-test
• Non-parametric tests
� Linear Regression• Standard curve
![Page 5: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/5.jpg)
GenEx Pro also includes modules for:
� Clustering• Hierarchical/Phylogenetics
� Principle component analyses• Principle component analysis
• Potential curve analysis
• Non-parametric tests
� Network analyses� Network analyses• Artificial neural network analysis
• Kohonen self organizing maps
� Linear Regression• Reverse calibration
![Page 6: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/6.jpg)
Absolute Quantification
5
10
15
20
25
30
0 1 2 3 4 5 6 7 8
??
![Page 7: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/7.jpg)
Standard Curve
20
25
30
Cq
5
10
15
20
0 1 2 3 4 5 6 7 8
log(conc)
![Page 8: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/8.jpg)
Residual plot
![Page 9: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/9.jpg)
Normal distribution = Gaussian distribution
![Page 10: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/10.jpg)
Outlier detection of repeats
• If at least three repeats are available, the data can be tested for
outliers with the Grubbs’ test.
)Cq(SD/)CqCq(Cqas −=• Autoscale data:
N 90% 95% 99%
3 1.15 1.15 1.15
4 1.46 1.48 1.50
5 1.67 1.72 1.76
6 1.82 1.89 1.97
7 1.94 2.02 2.14
8 2.03 2.13 2.27
9 2.11 2.22 2.39
10 2.18 2.29 2.48
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Prediction
25
30
Error bars are horizontal
The prediction band
is different than the
confidence band
Cq
5
10
15
20
0 1 2 3 4 5 6 7 8
Uncertainty increases
towards the edges
log(conc)
![Page 12: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/12.jpg)
Relative Quantification
?
A B
?
![Page 13: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/13.jpg)
Experimental Design
Subjects
Biological
samples
control treated
…….
RT samples*
PCR samples
*RT is the main source of variance. Therefore three replicates are built for
each biological sample to assure more precise mean estimate.
![Page 14: GenEx qPCR Data Analysis 2009-03-10 - Gene-Quantification · GenEx Standard includes modules for: Data editor • Data corrections • Normalizations • Scaling Data manager •](https://reader031.vdocument.in/reader031/viewer/2022011817/5e7c0154af903d6485546fb3/html5/thumbnails/14.jpg)
Statistical Analysis & Visualization Tools
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Multivariate Analysis
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Scatter plot
S3
S5 S4
S1 S2
S3
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Scatter plot
Cluster A
Cluster B
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Dendrogram
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Summary
� qPCR Data Analysis• Plan your experiments ahead of time
• Use replicates to evaluate variation
� GenEx Software Tool• Fully functional one-month free trial
• www.multid.se
• www.qpcrforum.com
� Come to MultiD’s booth in room S2 and ask us about:� Come to MultiD’s booth in room S2 and ask us about:• Software support
• Biostatistics training
• Data analysis consulting
• Research collaborations (SmartHEALTH, CD-Medics, ...)
Email: [email protected]
Phone: +46 733 145252