Assessment of differential expression of oncogenes in gastric adenocarcinoma by fluorescent multiplex RT-PCR assay

We screened samples of tumour and peripheral normal tissue for differential expression of oncogenes by using an approach of detecting the differences in expression of a number of oncogenes simultaneously. Total RNA was isolat ed from 29 pairs of normal and tumour tissue samples from patients with gas tric adenocarcinoma. Seven pairs of primers for oncogenes most probably ass ociated with the process of carcinogenesis in stomach including cyclin E, e rbB-3, HGR, c-met, TDGF/cripto, FGF-4, and EGF were used for the constructi on of fluorescent multiplex RT-PCR. Sense primers were 5' end-labelled with a fluorescent dye. 5-7 gastric oncogenes were simultaneously analysed for overexpression. Multiplex reverse transcription with a set of unlabeled pri mers was followed by a PCR reaction by adding the corresponding set of fluo rescent labelled PCR-primers. Expression of oncogenes was compared to GAPDH internal standard. Multiplex fluorescent RT-PCR results were analysed by c apillary electrophoresis on ABI-PRISM 310 Genetic Analyzer. Differential ex pression of oncogene mRNAs in tumour and normal tissue was assessed by comp arison of oncogene/GAPDH ratios in tumours and their peripheral normal muco sa. Our results show, that in most patients, comparing to normal tissue, we could estimate overexpression of at least one oncogene in a sample.