title: Droplet digital PCR quantifies host inflammatory transcripts in feces reliably and reproducibly.
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 a 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.
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Stauber
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors.forename: Nurmohammad
authors.initials: N
authors.lastname: Shaikh
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors.forename: M Isabel
authors.initials: MI
authors.lastname: Ordiz
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors.forename: Phillip I
authors.initials: PI
authors.lastname: Tarr
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, United States. Electronic address: manary@kids.wustl.edu.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Manary
mesh_terms: D002675:Child, Preschool; D005243:Feces; D006801:Humans; D007223:Infant; D007249:Inflammation / Q000175:diagnosis / Q000235:genetics*; D018836:Inflammation Mediators / Q000032:analysis*; D007413:Intestinal Mucosa / Q000502:physiology*; D008295:Malawi; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D018836:Inflammation Mediators; D012333:RNA, Messenger
keywords: Environmental enteric dysfunction; Gut inflammation; Human mRNA; Quantitative PCR; Stool; Stunting