Detection of Low-concentration Host mRNA Transcripts in Malawian Children at Risk for Environmental Enteropathy

Background and Objective: Transcriptomic analysis of fecal samples is an emerging method for the diagnosis of gastrointestinal pathology because it is noninvasive and requires minute volumes of analyte; however, detection of mRNA in low copy numbers in human stool is challenging. Our objective was to develop a method for detecting human mRNA suggestive of environmental enteropathy (EE) in feces. Methods: Stool samples from 70 Malawian children, 34 without EE and 36 with EE, as defined by dual sugar absorption, were used to develop the methodology for mRNA detection. Multiple RNA isolation techniques and polymerase chain reaction formats were tested to detect 38 potential mRNA biomarkers suggestive of EE, and the results compared. Results: RNA isolation using magnetic bead extraction best recovered host mRNA in stool, and digital droplet polymerase chain reaction was the most sensitive format to detect low copy numbers of mRNA. In all of the 70 samples, >20 copies of glyceraldehyde-3-phosphate dehydrogenase/200 mg of stool were detected. Copy numbers of potential biomarkers were normalized to glyceraldehyde-3-phosphate dehydrogenase, to account for interspecimen differences in concentration of human mRNA. Of the 38 transcripts chosen for initial evaluation, 24 had copy numbers >10 in all of the samples tested. Of the 6 potential markers measured in all of the 70 samples, REG4 best differentiated children with and without EE. Conclusions: A reproducible and reliable method to quantify human mRNA in stool present in low copy numbers has been developed, and may prove useful in investigations of EE and possibly other inflammatory gut conditions.