Andreas Pahl, Uwe Oelmller,* and Susanne Ullmann*
Department of Pharmacology, University of Erlangen, Erlangen, Germany

* QIAGEN GmbH, Hilden Germany Drug library screening based on gene expression analysis requires reliable and reproducible methods of RNA purification and sensitive, quantitative, real-time RT-PCR analysis. This article demonstrates how RNeasy 96 technology, automated on the BioRobot Gene Expression workstation, enabled reproducible, high-throughput sample preparation. One-step, real-time RT-PCR using the QuantiTect Probe RT-PCR Kit provided highly sensitive gene expression analysis to identify potential drugs to fight asthma.

Bronchial asthma is one of the commonest chronic diseases in modern society and the most common chronic childhood disease. The pathogenesis and causes of asthma are still poorly understood. Nonetheless, our knowledge of asthma pathogenesis has increased greatly over the past two decades, partially through recognition of the critical role played by cytokines in the inflammatory response.

Many cytokines are involved in asthma. Although some of these cytokines are involved in many inflammatory diseases, others are more specific to allergic inflammation, such as asthma. These cytokines include interleukins (IL) IL-4 and IL-13, which are derived from T-helper 2 (Th2) cells. Th1 cells, which are involved in cellular immunity, secrete other cytokines, such as IL-2. Gene expression analysis of these cytokines provides valuable information about the effects of potential drugs for asthma.

The goal of our research is to identify potential new drugs that selectively 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. Expression 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 identification of specific potential drugs to combat asthma.

Reference
1. Pahl, A. (2002) Manuscript in preparation.




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