TaqMan Gene Expression Arrays for Accurate Quantification of Toxicity Targets
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TaqMan Gene Expression Arrays for Accurate Quantification of Toxicity Targets
description
TaqMan Gene Expression Arrays for Accurate Quantification of Toxicity Targets. Agenda. Test Procedure Specifications Validation Results. Test Procedure. RNA isolation cDNA generation (may be skipped with one-step RT-PCR) Card loading and sealing Thermal cycling Data Analysis. - PowerPoint PPT Presentation
Transcript of TaqMan Gene Expression Arrays for Accurate Quantification of Toxicity Targets
TaqMan Gene Expression Arrays for Accurate
Quantification of Toxicity Targets
Agenda• Test Procedure
• Specifications
• Validation Results
Test Procedure• RNA isolation
• cDNA generation (may be skipped with one-step RT-PCR)
• Card loading and sealing
• Thermal cycling
• Data Analysis
Dynamic Range in the Cytokine Card
R2 = 0.9983
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0.0000010.000010.00010.0010.010.11
Log(fold concentration)
dd
Ct
average dCt
average dCt w/ t-test
median dCt
Log. (average dCt)
Dynamic Range in the Cytokine Card
y = 0.9643x - 0.0608
R2 = 0.9981
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Actual ddCt
Exp
ecte
d d
dC
t
Average plus 6 Stdev
is Fold Discrimination
Calculate Average of 40 RQ values
Normalize RQ values to positive fold
Calculate RelQuant Values
(20 for each sample--40 total)
Calculate dCt Calibrator Calculate dCT sample 1 Calculate dCt sample 2
3 cytokine cards with 20 plasmid targets
Fold-Discrimination in the Cytokine Card
Specifications: Relative Quantification (median/ttest) for 3 Cytokine Cards
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Spec
Avg + 6xStdev = 3.79
Avg 1.34Stdev 0.41
DNAx: Cytokine Gene Expression and PBMC Activation
Source Precision Medicine: Cytokine Gene Expression and PBMC Activation
no stimulation
condition A
condition B
condition C
LegendTarget Dye: FAM
Endogenous Control Dye: VIC
Calibrator Sample: no stimulation
Not enough signal to calculate ²Ct reliably
Value displayed is a minimum
Value displayed is a maximum
Phase 1 Molecular Toxicology: Cytokine Gene Expression in Liver Cell Cultures
Control
Actinomycin D
Legend
Phase 1 Molecular Toxicology: MMC Incubations of Liver Cells in the Human Toxicology Card
Phase 1 Molecular Toxicology: MMS Incubations of Liver Cells in the Human Toxicology Card
Phase 1 Molecular Toxicology: Carnustine Incubations of Liver Cells in the Human Toxicology Card
Phase 1 Molecular Toxicology: Cytokine Gene Expression in RNA Isolated from Blood Cells
Control
3-1F-33-Cau
1-1F-28-Cau
2-1M-46-Cau
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Phase 1 Molecular Toxicology: Toxicology Gene Expression in RNA Isolated from Blood Cells
7-1F40s
5-1M50
6-1F50
Control
1-1F28
Legend
Phase 1 Molecular Toxicology: Cytokine Gene Expression in RNA Isolated from Lymphocyte Cultures
Control
Pooled
PBL
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Phase 1 Molecular Toxicology: Toxicology Gene Expression in RNA Isolated from Lymphocyte Cultures
Control
Pooled
PBL
Legend
