PubMed · 8484039
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M Vázquez Caruncho, J L Díaz Valiño, J C Gallego Ojea. 1993. [Colonic tuberculosis].. https://pubmed.ncbi.nlm.nih.gov/8484039/
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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 microgram 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.
To investigate the effects of PACAP-27 on electrolyte transport across the isolated human intestinal mucosa, changes in short-circuit current (Isc) were measured in Ussing chamber experiments. Serosally added PACAP-27 increased Isc in a concentration-dependent manner, eliciting a similar maximal effect in both the jejunal and the colonic mucosa. Bumetanide inhibited Isc responses, indicating stimulation of Cl- secretion. The potency and efficacy of PACAP-27 were comparable to those of VIP, suggesting that both peptides activate intestinal secretion by way of a common receptor located in the basolateral membrane of the intestinal epithelium.