Extracting Fecal RNA for miRNA Analysis

The extraction of total RNA from feces was based on the method described by Reck et al.^{51 (link)}, and we used commercially available kits to isolate total RNA from small amounts of stool samples (PowerMicrobiome™ RNA Isolation Kit, MoBio Laboratories, Germany). In short, approximately 0.25 g of feces was transferred to a glass bead tube (0.1 mm). Shortly before the feces sample was completely thawed on ice, added β-mercaptoethanol to the MoBio lysis buffer to make the final concentration of 10 μl/ml in the tube, and homogenized on the Vortex-Genie 2 Mixer at the highest speed for 10 min. The sample was centrifuge at 13,000×g for 1 min at room temperature, and further steps included DNAse I treatment of the samples and elution of RNA in nuclease-free water, which were performed according to the manufacturer's instructions. The RNA concentration was measured using Nanodrop ND 2000 (Thermo Scientific, USA).
The miRNA profiles in human feces were determined by NanoString nCounter System (NanoString Technologies) and the nCounter Human-v2 miRNA Panel with ~ 800 unique miRNA barcodes. Assays and spike-in controls were used for normalization based on identical amounts of input RNA.