Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain.

We constructed a plasmid for the in vitro synthesis of a competitor RNA for use as an internal exogenous control during reverse transcriptase--PCR (RT-PCR) detection of epidermal growth factor receptor (EGFR) expression.The competitor RNA harbors a 32-base deletion compared with wild-type EGFR mRNA and generates a PCR product that is easily distinguished from the wild-type PCR product by agarose gel electrophoresis.We encountered the problem of heteroduplex formation during later stages of PCR, which could be solved by decreasing the PCR cycle number.This was accompanied by a significant loss of sensitivity.Sensitivity could be restored by using a novel and extremely sensitive DNA stain (SYBR Green I) instead of ethidlum bromide.C o m p e t i t i v e reverse transcriptase-polymerase chain reaction (competitive RT-PCR) is being used increasingly for determination and quantification of gene expression, particulary when test sample size is limited and high sensitiv-ity is required.(1,2~ The use of internal control templates in competitive RT-PCR is necessary to ensure that the reverse transcription and the PCR have functioned as expected and to generate a standard curve from which the concen-FIGURE 1 SYBR Green I-(A) and ethidium bromide-stained (B) agarose gel of competitive RT-PCR products (12 ~1 PCR product).(A) The extreme sensitivity of this DNA stain; (B) the effect of decreasing PCR cycle numbers on the formation of heteroduplexes.The total RNA (S00 ng) extracted from a breast cancer sample, was reverse transcribed and amplified in the presence of 50 pg of EGFR-competitor RNA.Lanes 20-30 correspond to 20--30 PCR cycles; (lane M): Bio-Marker Low (Bioventures, Inc., Murfreesboro, TN). PCR Methods andApplicutions