Calibrated user-friendly reverse transcriptase-PCR assay: Quantitation of epidermal growth factor receptor mRNA

Abstract

We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CURT- PCR, a calibration curve was developed by plotting the ratio between the amount of PCR product originating rom the calibrator and the RNA internal standard vs. the amount of EGFR mRNA present in the calibrator. A fixed amount of RNA internal standard was coamplified with the EGFR mRNA present in the calibrator in the sample, using the same primer set. The primers were chosen in regions of the EGFR mRNA that show 100% homology between human, rat, and mouse. The amount of EGFR in the unknown samples was calculated from the calibration curve based on the ratio between PCR product originating from the sample and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17). We conclude that the CURT-PCR method developed is suitable for routine application such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run and to accept or reject the results according to existing rules for quality assurance.