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Research Project: Pediatric Clinical Nutrition

Location: Children's Nutrition Research Center

Title: Droplet digital PCR quantifies host inflammatory transcripts in feces reliably and reproducibly

item STAUBER, JENNIFER - Washington University School Of Medicine
item SHAIKH, NURMOHAMMAD - Washington University School Of Medicine
item ORDIZ, M - Washington University School Of Medicine
item TARR, PHILLIP - Washington University School Of Medicine
item MANARY, MARK - Children'S Nutrition Research Center (CNRC)

Submitted to: Cellular Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2016
Publication Date: 5/1/2016
Citation: Stauber, J., Shaikh, N., Ordiz, M.I., Tarr, P.I., Manary, M.J. 2016. Droplet digital PCR quantifies host inflammatory transcripts in feces reliably and reproducibly. Cellular Immunology. 303:43-49.

Interpretive Summary: Biological material in human fecal samples are largely composed of bacteria, making it difficult to measure human biological compounds. Our lab has developed 2 new techniques which allow for such measurements, and this method is described and validated in this paper. These processes may provide a new perspective for researchers in the future.

Technical Abstract: The gut is the most extensive, interactive, and complex interface between the human host and the environment and therefore a critical site of immunological activity. Non-invasive methods to assess the host response in this organ are currently lacking. Feces are the available analyte which have been in proximity to the gut tissue. We applied a method of concentrating host transcripts from fecal specimens using an existing bead-based affinity separation method for nucleic acids and quantified transcripts using droplet digital PCR (ddPCR) to determine the copy numbers of a variety of key transcripts in the gut immune system. ddPCR compartmentalizes the reaction in a small aqueous droplet suspended in oil, and counts droplets as either fluorescent or non-fluorescent. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize transcript concentration. This method was applied to 799 fecal samples from rural Malawian children, and over 20,000 transcript concentrations were quantified. Host mRNA was detected in >99% samples, a threshold for target detection was established at an average expression of 0.02 copies target/GAPDH, above which correlation coefficient between duplicate measurements is >0.95. Quantities of transcript detected using ddPCR were greater than standard qPCR. Fecal sample preservation at the time of collection did not require immediate freezing or the addition of buffers or enzymes. Measurements of transcripts encoding immunoactive proteins correlated with a measure of gut inflammation in the study children, thereby substantiating their relevance. This method allows investigators to interrogate gene expression in the gut.