Exfoliated cells in stool: a source for reverse transcription-PCR-based analysis of biomarkers of gastrointestinal cancer

Abstract

Because the colonic mucosa is in direct contact with digesta, luminal exposure to potentially carcinogenic or chemopreventive agents may be important in colorectal carcinogenesis, independently of the effects of systemic exposure through the circulation. To date, few biomarkers for luminal exposure have been identified, and isolation of reasonably good quality fecal human RNA remains difficult. In this study, we assessed the yield and quality of RNA extracted from 10 human stool samples after storage with several commercially available preservatives compared with stool samples immediately frozen in liquid nitrogen. This study shows that careful design of primer pairs which amplify a short length of DNA is key to obtaining interpretable and reproducible results. Moreover, the use of commercially available RNA preservation kits enables investigators to collect usable fecal samples from large populations. Of all the preservative methods tested, RNAlater had the best performance in terms of overall quality, quantity, and level of genomic DNA contamination, and thus deserves further investigation.

Figure 1

RT-PCR product analysis on agarose gel. Ten microliters of the PCR product was electrophoresed on 3% agarose gels containing ethidium bromide. L, a 1 kb plus molecular DNA ladder (Invitrogen). H, human colon cell line HCT-116. 2 to 15, patient ID numbers. A. Expression of the second primer pair products (234 bp) with the 10 liquid nitrogen preserved samples. B. Expression of the third primer pair products (103 bp).