Real-time polymerase chain reaction quantitation of relative expression of genes modulated by p53 using SYBR Green I.
Real-time quantitative polymerase chain reaction (QPCR) using the Roche LightCycler was used to verify the expression of asparagine 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 beta-galactosidase as a control. After 24 h of infection, RNA was harvested and used for microarray analysis. ASNS was one of several genes whose expression was down-regulated by WT p53 and up-regulated in the presence of mutant p53. Expression levels of ASNS were 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. ASNS was found to be down-regulated in the presence of WT p53 and up-regulated by mutant p53.
Stagliano
Katherine E Roth
KE
Department of Biochemistry, Virginia Commonwealth University, Richmond, USA.
Carchman
Evie
E
Deb
Sumitra
S
D052160:Benzothiazoles; D045744:Cell Line, Tumor; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009930:Organic Chemicals / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D011804:Quinolines; D012984:Software; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D016159:Tumor Suppressor Protein p53; C098022:SYBR Green I