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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
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
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
-2
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
0 5 10 15 20 25 30 35 40
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
Legend
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
Legend
Phase 1 Molecular Toxicology: Toxicology Gene Expression in RNA Isolated from Lymphocyte Cultures
Control
Pooled
PBL
Legend
