A method for isolating and analyzing human mRNA from newborn stool

Abstract

Efforts to characterize the human transcriptome have largely been limited to blood, urine, and tissue analyses (i.e., normally sterile materials). We report here an extraction protocol using commercially available reagents to obtain high-yield, reverse-transcribable RNA from human stool. Quantitative reverse transcriptase polymerase chain reactions demonstrated minimal intra-specimen but considerable intra-subject variability over time of transcripts for interleukin-6 (IL-6), IL-8, epidermal growth factor (EGF), calprotectin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This technique now expands opportunities to use the human fecal transcriptome to characterize gastrointestinal pathophysiology.

MAPK-Activated Protein Kinase 2 Contributes to Clostridium difficile-Associated Inflammation

“…All of these patients had gastrointestinal symptoms. Inflammatory cytokine gene expression can be detected in fecal extracts from patients with other bacterial causes of enteritis (68,69). Inflammatory cytokines and lactoferrin are also elevated in the feces of patients with active inflammatory bowel disease (9,70,71).…”

Proinflammatory fecal mRNA and childhood bacterial enteric infections

“…5 Even if RNA degradation does occur in stool, as long as the mRNA of interest and the GAPDH transcripts degrade at the same rates, and sufficient non-degraded transcript (the calprotectin and GAPDH primers produce RNA amplicons that are 107 and 122 base pairs, respectively) remains, ratios can be determined. There are two additional advantages to determining mRNA abundance in stool, compared to assays of proteins.…”

“…Fecal mRNA has been underexplored as an analyte, probably because of reservations regarding its feasibility. However, recent reports 4,5 demonstrate that stool mRNA isolation and analysis is reproducible and useful in characterizing a wide array of host intestinal responses in children and adults. If fecal mRNA reflects pathophysiology within the digestive system, its analysis could open new vistas on gut biology in health and disease, and supplant protein determinations such as enzyme immunoassays (EIAs).…”

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

“…Human mRNA has been detected in stool samples (16)(17)(18)(19), and could provide a novel window on the host gut in EE. mRNA sequences specific for small intestine inflammation are likely to be present in small amounts of feces.…”