( pression of IL-2 mRNA was essen...)High-throughput gene expression analysis to screen for anti-asthma drugs

pression of IL-2 mRNA was essentially inhibited by treatment with CSA. Expression of Th2 specific IL-13 mRNA was reduced to a lesser extent in the CSA-treated cells, and IL-4 mRNA expression was the same in unstimulated cells and CSA-treated cells, indicating that the drug prevented the stimulation-dependent increase in gene expression. Expression of -actin mRNA, used as a housekeeping transcript control, remained unchanged.

High-Throughput Screening of Cytokine Expression Figure 1 PBMCs were stimulated with TPA/lonomycin or unstimulated, and treated with CSA or not treated, as indicated (see Materials and methods). Total RNA was isolated using RNeasy 96 technology, fully automated on the BioRobot Gene Expression workstation. Real-time, quantitative, one-step RT-PCR was carried out in a 384-well format using the QuantiTect Probe RT-PCR Kit and primers and probes specific for the transcripts indicated. All reactions were carried out with 2 to 4 replicates. Threshold cycles are indicated. Conclusions

• Fully automated RNeasy 96 technology on the BioRobot Gene Expression workstation enabled walkaway operation for high throughput drug screening applications.
  
• Quantitative, real-time, one-step RT-PCR using the QuantiTect Probe RT-PCR Kit provided highly sensitive results to analyze expression of different cytokines in response to drug candidates.
  
• High-throughput drug screening using these technologies enables identific
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