Real-time quantitative polymerase chain reaction (QPCR) using the Roche LightCycler� was used to verify the expression ofasparagine synthetase (ASNS)identified by microarray analysis as a target of p53 transrepression and mutant p53 transactivation. A p53-null cell line derived from lung carcinoma, H1299, was infected with recombinant adenovirus expressing wild-type (WT) p53, mutant p53-D281G, or β-galactosidase as a control. After 24 h of infection, RNA was harvested and used for microarray analysis.ASNSwas one of several genes whose expression was down-regulated by WT p53 and up-regulated in the presence of mutant p53. Expression levels ofASNSwere measured relative to an exogenously applied quality-control nucleic acid template. Real-time PCR product accumulation was monitored using the intercalating dye, SYBR� Green I, which exhibits a higher fluorescence upon binding of double-stranded DNA. Relative gene expression was calculated using conditions at the early stages of PCR, when amplification was logarithmic and, thus, could be correlated to initial copy number of gene transcripts.ASNSwas found to be down-regulated in the presence of WT p53 and up-regulated by mutant p53.
