RNA-based drug screening using automated RNA purification and real-time RT-PCR1.
A new system has been developed for RNA-based drug screening, and the feasibility of this approach has been demonstrated by the identification of new immunomodulating compounds. Peripheral blood mononuclear cells were chosen as the cellular assay system. Cells were either stimulated by TPA/ionomycin to produce T cell cytokines as asthma targets or stimulated by lipopolysaccharide to produce proinflammatory cytokines as targets for chronic obstructive pulmonary disease (COPD). The authors developed a new fully automated system for RNA purification from cells grown in 96-well plates. Gene expression was determined in 384-well plates using real-time quantitative one-tube RT-PCR. Small interdonor variation could be demonstrated. The assay system was validated with known immunosuppressants cyclosporine and dexamethasone. Screening of 800 compounds resulted in 9.5% compounds inhibiting the induction of at least 1 T cell derived cytokine and 6.8% compounds inhibiting at least 1 cytokine relevant for COPD. All these compounds were retested by analyzing remaining RNA from the 1st round of screening. The reproducibility of hits was between 56% and 74% for different cytokines. One compound selectively inhibited TNF, which was confirmed by IC(50) determination. Analyzing its effect on cells from different donors revealed little interdonor variation. In conclusion, the authors established fully automated RNA isolation and precise gene expression profiling using real-time RT-PCR for drug screening.
Ullmann
Susanne
S
QIAGEN, Hilden, Germany.
Hage
Thorsten
T
Draheim
Regina
R
Egerland
Ute
U
Oelmüller
Uwe
U
Brune
Kay
K
Pahl
Andreas
A
D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D020869:Gene Expression Profiling; D006801:Humans; D009363:Neoplasm Proteins / Q000737:chemistry; D009841:Oligonucleotides; D029424:Pulmonary Disease, Chronic Obstructive / Q000097:blood; D012313:RNA / Q000097:blood / Q000302:isolation & purification*; D047889:Receptors, Tumor Necrosis Factor, Type II; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D053319:Tumor Necrosis Factor Decoy Receptors
D016207:Cytokines; D009363:Neoplasm Proteins; D009841:Oligonucleotides; D047889:Receptors, Tumor Necrosis Factor, Type II; D053319:Tumor Necrosis Factor Decoy Receptors; D012313:RNA