Noninvasive detection of putative biomarkers for colon cancer using fecal messenger RNA.
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abstract
Deaths from colon cancer number over 60,000 each year in the United States. Because early detection results in a high cure rate, development of noninvasive techniques for detection of colon cancer has received much interest. The ability to detect early changes in colonocyte genes and gene expression would provide valuable information. We have shown previously that alterations in protein kinase C (PKC) isoform expression are associated with changes in colonic cell proliferation, a key intermediate marker for the prediction of tumorigenesis. Here, we describe a method for the quantitative detection of mRNAs for select PKC isoforms isolated from rat feces containing exfoliated colonocytes. After total RNA extraction from fresh fecal material, polyadenylated RNA was selectively purified and quantitated with slot blotting and hybridization to oligodeoxythymidylic acid. Fecal polyadenylated RNA was used for semiquantitative (mimic) RT-PCR to quantitate PKC isoform mRNA expression. We detected mRNA for PKC-alpha, PKC-delta, PKC-epsilon, and PKC-sigma, but not for PKC-beta or PKC-gamma, which is consistent with the profile of isoforms detected previously in scraped colonic mucosa using immunoblot analysis. This noninvasive method, utilizing feces containing exfoliated colonocytes, is a sensitive noninvasive technique for quantitating luminal mRNAs. This provides a means to monitor gene expression of colonic epithelial cells, which may have predictive value in monitoring the neoplastic process.
