Separation and quantitation of reverse transcriptase polymerase chain reaction fragments of basic fibroblast growth...

In human ovarian carcinomas (epithelial and endometrial tumors) the presence of mRNA coding for the basic fibroblast growth factor (bFGF) was previously demonstrated by reverse transcription-polymerase chain reaction (PCR). For quantitation purposes, competitive PCR is adopted, using a competing fragment a sequence of a highly homologous bovine bFGF. However, separation and detection of the PCR products by slab gel electrophoresis and ethidium bromide staining gives poor quantitative data due to the nonstoichiometric binding of the dye. Thus, the only possible quantitation that can be obtained is via autoradiography with 32P-labeled primers. We report here a capillary electrophoresis protocol, in 6% linear, liquid polyacrylamide as a sieving system, able to fully resolve and quantify the undigested (354 bp) bovine and the digested (295 bp and 59 bp) human fragments, with peak ratios in good agreement with the autoradiographic data.