Rapid detection of apoptosis through real-time reverse transcriptase polymerase chain reaction measurement of the small cytoplasmic RNA Y1.
Apoptosis could be measured in mammalian cells by measuring the degradation of the small cytoplasmic human RNA Y1 (hY1) by real-time quantitative fluorescent reverse transcriptase polymerase chain reaction (RT-PCR). In FAS-antibody-treated Jurkat T cell leukemia cells degradation of hY1 occurred rapidly and was complete at about 6h. As in apoptotic Jurkat cells, protein synthesis is arrested only after about 12h; this implies that protein synthesis can occur without scRNA-Y1. The degradation of hY1 could be blocked with peptide-based inhibitors of caspase 8 and with lower efficacy with caspases 1 and 3 and with ZnSO4. No effects were observed after inhibition of caspases 2, 6, and 9. Degradation of hY1 could also be demonstrated after treatment of A549 lung carcinoma cells treated with Staurosporin, Paclitaxel, or the histone deacetylase inhibitor LAQ824. RT-PCR systems based on SYBR Green, Amplifluor Uniprimer, or 5' nuclease Taqman could be used with increasing sensitivity. This apoptosis assay requires quantities of total cell RNA equivalent to only a few tissue culture cells and is especially suited to measure apoptosis in projects where RNA samples are already available from gene expression studies.
Asselbergs
Fred A M
FA
Department of Functional Genomics, Novartis Pharma AG, WSJ-360.601, CH-4002 Basel, Switzerland. fred_am.asselbergs@pharma.novartis.com
Widmer
Roland
R
D017209:Apoptosis / Q000235:genetics*; D015415:Biomarkers / Q000032:analysis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D020871:RNA Stability; D020733:RNA, Small Cytoplasmic / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019014:fas Receptor / Q000378:metabolism
D015415:Biomarkers; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D020733:RNA, Small Cytoplasmic; D019014:fas Receptor; D020169:Caspases