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

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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

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3.50

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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