Quantification of c-erbB-2 gene expression in breast cancer by competitive RT-PCR

We adapted competitive reverse transcription-polymerase chain reaction (RT-PCR) for quantitative evaluation of c-erbB-2 expression in breast cancer. A fixed amount of cDNA target was coamplified with dilutions of a nonhomologous DNA sequence used as an internal standard (IS). The IS and the target shared the same primer sequences but yielded PCR products of different sizes (348 and 340 bp, respectively). A fluorescent sense primer was used so that the PCR products separated on denaturing polyacrylamide gels could be quantified with an automated DNA sequencer. In human breast cancer cell lines, c-erbB-2 expression was found to range from 0.151 to 652 zmol/μg total RNA (i.e., 91 to 391 200 molecules/μg total RNA; 1 zmol = 10−3 amol), with the two highest values corresponding to the c-erbB-2 overexpressing cell lines MDA-MB-453 and SK-BR-3. In a series of 39 breast cancer biopsies, the concentrations ranged from 0.117 zmol/μg to 1.15 amol/μg total RNA (i.e., 70 to 690 000 molecules/μg total RNA). The c-erbB-2 oncoprotein (p185) was determined in 30 samples by an enzyme immunoassay. A close correlation was found between c-erbB-2 gene and oncoprotein expression (P = 0.0067). Thus, this competitive RT-PCR method appears to be a reliable way to evaluate the expression of c-erbB-2 in small tumor samples.