( inhibit expression of specific cyt...)High-throughput gene expression analysis to screen for anti-asthma drugs

inhibit expression of specific cytokines. High-throughput RNeasy 96 RNA isolation technology automated on the BioRobot Gene Expression workstation and real-time RT-PCR analysis using the QuantiTect Probe RT-PCR Kit enable
sensitive analysis of cytokine gene expression. These technologies speed the progresstoward new specific drugs to fight asthma.

Materials and methods

Peripheral blood monocytes (PBMCs) were grown in 96-well plates with 200,000 cells per well. The cells were treated with different drug candidates for 30 minutes. TPA/ Ionomycin was then added to stimulate Th1 and Th2 cells, and the cells were incubated for a further 4 hours. Unstimulated cells were used as a control for basal gene expression levels. The cell cultures were then placed on the BioRobot Gene Expression workstation for fully automated RNA purification using the RNeasy 96 BioRobot 8000 Kit. Quantitative, real-time RT-PCR of cytokine transcripts was carried out in 384-well format on the ABI PRISM 7900HT Sequence Detection System using the QuantiTect Probe RT-PCR Kit.

Results and discussion

Automated RNA isolation on the BioRobot Gene Expression workstation and quantification by one-step, real-time QuantiTect Probe RT-PCR enable creening of large numbers of synthetic and natural substances for their effects on cytokine expression in cells. We have used this approach to screen for asthma drugs that inhibit expression of Th2-specific cytokines (1). Figure 1 shows an example using cyclosporine A (CSA), which was used as a control compound during the screening process. CSA is an immunosuppressive agent known to inhibit transcription of IL-2 and used to prevent organ transplant rejection. Ex
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