The mammalian gut is a busy site where digestion, immune surveillance, and host-microbe interactions take place. Intestinal microRNAs—small, non-coding molecules that regulate gene expression—participate in many of these processes.1 These microRNAs are detectable in the feces, offering a non-invasive tool to study gut health.2
Fascinated by the big impact of these small molecules, microbiologist Emma Layton wanted to study fecal microRNAs as part of her graduate studies at The University of Manchester. However, she ran into some trouble. “There isn't anyone that's already said, ‘This is how you should extract RNA from fecal samples’, [or] ‘This is the best way to build the libraries for small RNA sequencing’,” said Layton, who is now at the University of Oxford.
To bridge this gap, Layton and her colleagues recently optimized a pipeline for fecal microRNA detection and demonstrated its ability to profile the molecules in stool samples from mice.3 Their findings, published in Nature Communications, provide a robust framework to isolate and sequence fecal microRNAs that can serve as biomarkers of diseases like cancer.
“I found [this study] very interesting,” said Alessio Naccarati, a molecular epidemiologist studying fecal miRNAs at the Italian Institute for Genomic Medicine (IIGM), who was not involved in the study. This research could have important implications for diagnostic analysis, he added.
Previously, researchers isolated and sequenced microRNAs using a variety of methods. To standardize a pipeline, Layton and her team tested and tweaked various parts of these protocols. They identified an ideal temperature to store fecal pellets to produce high-quality RNA and the PCR settings to build a suitable cDNA library for RNA sequencing. Compared to three other commonly used methods, their process yielded the most microRNA reads and more microRNA species. “When I saw that we'd actually improved the efficiency and got the highest number of microRNA reads, I was surprised and really, really happy,” said Layton.
Equipped with an optimized protocol, Layton and her team evaluated its efficacy in profiling microRNAs expressed during intestinal infections. They infected mice with the whipworm Trichuris muris, an intestinal parasite, and profiled their fecal microRNA.4
Compared to control animals, infected mice expressed higher levels of three microRNAs, including miR-200c, and lower expression of four, including miR-29a. Identifying the predicted targets of these microRNAs indicated that the molecules could potentially influence fibrosis, where excessive production of collagen by fibroblasts leads to scarring. Other conditions like inflammatory bowel disease cause similar scarring, opening the possibility of using microRNAs as fibrosis biomarkers.
To study the role of these microRNAs in fibrosis, the researchers treated cultured fibroblasts with molecules mimicking or inhibiting these microRNAs. Fibroblasts exposed to a miR-29a inhibitor produced more collagen, indicating that reduced miR-29a after parasitic infection promoted collagen deposition and fibrosis. Conversely, adding a miR-200c mimic increased collagen production in fibroblasts, consistent with higher miR-200c levels post infection promoting fibrosis.
Finally, Layton and her team investigated this observation in vivo by studying the pathology of intestinal tissue from control and T. muris-infected mice. They observed extensive fibrosis in the latter, where they had also detected more fibrosis-associated microRNAs, confirming their prediction of these small RNAs being involved in infection-induced fibrosis. Seeing the in vivo results was a nice validation of the technique’s potential to identify disease biomarkers, said Layton.
“[That] they were able to demonstrate that [infection-associated changes] are connected with microRNAs is probably the most important and novel thing,” said Naccarati. More studies under different conditions could help researchers better understand how well this protocol works to identify disease biomarkers, he added.
The researchers described the framework “very well,” said Barbara Pardini, a molecular epidemiologist studying fecal miRNAs at IIGM, who was not involved in the study. “But on the other side, it could be costly.”
Layton said that future work could focus on making this pipeline high throughput and more cost-efficient. “I think there's definitely a lot of optimizations to the technique itself that that can still be made, and hopefully this little piece of work will inform that.”
- Runtsch MC, et al. MicroRNAs and the regulation of intestinal homeostasis. Front Genet. 2014;1;5:347.
- Rashid H, et al. Fecal microRNAs as potential biomarkers for screening and diagnosis of intestinal diseases. Front Mol Biosci. 2020;7:181.
- Layton E, et al. An optimized fecal microRNA sequencing pipeline reveals fibrosis in Trichuris muris infection. Nat Commun. 2025;16(1):1589.
- Else KJ, et al. Whipworm and roundworm infections. Nat Rev Dis Primers. 2020;6(1):44.
