Real-time polymerase chain reaction is a sensitive and precise method for quantifying transcription from reporter plasmids.
Sensitive methods are required for studying promoter activity of weakly expressed genes using reporter systems. In this study it is shown that the sensitivity of the traditional enzymatic assay for measuring LacZ activity is too low when studying the promoter activity of epiregulin, a member of the epidermal growth factor (EGF) system. Consequently, a new real-time polymerase chain reaction (PCR) method was developed to evaluate the expression of the reporter gene LacZ. This method can be used for monitoring promoter activity of weakly expressed genes. T24A cells with LacZ expression driven by the cytomegalovirus (CMV) promoter were used to compare real-time PCR and the traditional enzymatic detection system. The real-time PCR method had a more than 1000-fold higher sensitivity than the enzymatic assay. LacZ mRNA could be quantified from as few as two cells with an imprecision of 19%. The increased sensitivity enabled the measurement of LacZ mRNA transcription driven by the epiregulin promoter in stably transfected T24A bladder cancer cells. This allowed a reproducible quantification of a 3-fold transcriptional increase from the epiregulin promoter caused by serum stimulation of stably transfected T24A cells. Clearly, this new method is well suited to quantify relatively small changes in transcriptional activity from weakly expressed genes.
Ornskov
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D
Department of Clinical Biochemistry, Aarhus Sygehus, University Hospital in Aarhus, Denmark.
Nexo
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E
Sorensen
B S
BS
D045744:Cell Line, Tumor; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection