The expression of growth factor receptor messenger RNA is difficult to quantitate due to low copy number. We describe a quantitative polymerase chain reaction that rapidly, reproducibly assays expression of human insulin-like growth factor I (IGF-I) receptor mRNA from small, biopsy-sized, specimens of tissue. This was then clinically applied to surgically resected specimens of colon. Total RNA was isolated from normal colonic mucosa and documented tumors from 4 patients undergoing resection. The mRNA was first reverse-transcribed with an oligomer bearing a complementary sequence specific for the mRNA at its 3' end, and a sequence complementary to an intervening intron of the IGF-I receptor gene at the 5' end. Competitive PCR was then performed in the presence of the cDNA product and exogenously added genomic DNA, with an upstream primer complementary to the exon sequence of the gene of interest and a downstream primer complementary to the intron sequence that was tagged to the cDNA. The genomic DNA was used as the internal standard. To calculate the number of copies of mRNA per μg total RNA, a standard curve was used. No difference was noted in IGF-I receptor expression between neoplastic and normal colonic mucosa. This quantitative PCR is accurate, rapid, and requires very small amounts of tissue. Potential uses are in determining genetic expression of growth factor receptors or putative tumor markers preoperatively from small samples obtained during diagnostic colonoscopy or from samples obtained at surgery.
ASJC Scopus subject areas
- Cancer Research