Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces Jordan M. Nechvatal a , Jeffrey L. Ram a, ⁎ , Marc D. Basson b,c,d , Phanramphoei Namprachan a , Stephanie R. Niec a , Kawsar Z. Badsha e , Larry H. Matherly d,f , Adhip P.N. Majumdar h , Ikuko Kato d,g a Department of Physiology, Wayne State University, Detroit, MI, USA b Surgical Service, John D. Dingell VA Medical Center, Detroit, MI, USA c Department of Surgery, Wayne State University, Detroit, MI, USA d Karmanos Cancer Institute, Detroit, MI, USA e Department of Nutrition and Food Science, Wayne State University, Detroit, MI, USA f Department of Pharmacology, Wayne State University, Detroit, MI, USA g Department of Pathology, Wayne State University, Detroit, MI, USA h Department of Internal Medicine, Wayne State University, Detroit, MI, USA Received 14 August 2007; received in revised form 26 October 2007; accepted 13 November 2007 Available online 21 November 2007 Abstract Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34% ± 9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers. © 2007 Elsevier B.V. All rights reserved. Keywords: Bacteroides; DNA extraction; DNA preservation; Enteric bacteria; Feces; Stool 1. Introduction Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastro- intestinal tract health. For example, bacteria activate or me- tabolize potential carcinogens (Blaut et al., 2006; Knasmuller et al., 2001; Vanhaecke et al., 2006) or can have anti-tumor effects (Fukui et al., 2001) that may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. With the gastrointestinal tract being the largest area of the body that is constantly exposed to ingested/digested food and microorganisms, it is conceivable that luminal exposure may play a significant role in the development of colorectal cancer. Available online at www.sciencedirect.com Journal of Microbiological Methods 72 (2008) 124 – 132 www.elsevier.com/locate/jmicmeth ⁎ Corresponding author. Department of Physiology, Wayne State University, 540 E. Canfield Avenue, Detroit, MI 48201 USA. Tel.: +1 313 577 1558; fax: +1 313 577 5494. E-mail address: jeffram@med.wayne.edu (J.L. Ram). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.11.007