TurboCapture 384 mRNA Kit (5)

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Gene Expression Profiling Using L1000 Platform: HPTECs cultured in 96-well plates were treated with the 10 toxic and nontoxic compounds in the discovery panel (Supplemental Table 1) in six concentrations (1.6 µM to 20 mM) for 3, 6, 12 and 24 h, and lysed with 100 µl of TCL buffer (Qiagen). Cell lysates were added to the well of a TurboCapture 384 plate to perform mRNA isolation and cDNA synthesis in the same well (TurboCapture 384 mRNA kit; Qiagen). High throughput gene expression analysis is based on the measurement of about 1000 transcripts as described previously.40 Replicates were averaged and values were z-scored across all data. For each pair of gene and drug, Spearman’s correlation Rho1, …, Rho4 between the gene expression and the six doses across all four time points (t1, …, t4) was calculated. The sum of squares of correlation | Rho|2=Σ Rhoi2 for i=1, …, 4 if Rhoi>0 was compared with values of | Rho|2 obtained from the correlation of six random values. The comparison with 5,000,000 random values of | Rho|2 yields an empirical P value. A FDR value was obtained using the Benjamini and Hochberg procedure and gene expression was considered to be significantly positively correlated with drug if FDR<0.01.

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