A real-time one-step reverse transcriptase-polymerase chain reaction method to quantify c-erbB-2 expression in human breast cancer

We developed a real-time one-step reverse transcriptase-polymerase chain re action (RT-PCR) method for the routine quantification of c-erbB-2 oncogene expression in breast cancer, using a 7700 ABI PRISM Sequence Detector Syste m (Perkin Elmer-Applied Biosystems, Courtaboeuf, France). The real-time qua ntification of the polymerase chain reaction products is based on the TaqMa n 5' nuclease assay. The optimal experimental conditions we determined were as follows: 6 mM MgCl2, 200 nM of fluorogenic probe, 200 nM of each primer , and 12.5 units MuLV reverse transcriptase. The GAPDH housekeeping gene wa s used for normalization of c-erbB-2 expression. In human breast cancer cel l lines, the normalized expression of c-erbB-2 ranged from 8 x 10(-6) to 2, 600 x 10(-6), the two highest values corresponding to the c-erbB-2 overexpr essing cells MDA-MB-453 and SK-BR3. In a series of 100 breast cancer sample s, c-erbB-2 normalized expression was found to range from 0.4 x 10(-6) to 3 50 x 10(-6). A close correlation was observed between this real-time one-st ep quantitative RT-PCR method and both semiquantitative conventional RT-PCR (N = 22; r = 0.8543; P < .0001) and c-erbB-2 protein expression (p185) qua ntified by an enzyme immunoassay (EIA) (N = 27; r = 0.71; P <.0001). The cu rrent real-time RT-PCR assay is rapid, sensitive, and reproducible and appe ars particularly suitable to quantify gene expression in large series of sa mples.