Total RNA Extraction from Stool Samples

Total RNA (including miRNAs) from fresh stool samples was extracted using the TRIzol method according to the manufacturer’s instructions, and minor modifications were applied (TRIzol, Invitrogen, Waltham, MA, USA). A similar amount of stool was homogenized with the TRIzol reagent, and the aqueous phase was collected in a new collection tube after centrifugation. This procedure was repeated 3 times. In the first round, 0.2 mL of chloroform was added; in the second wash, 0.5 mL of isopropanol was added; and in the third wash, 1 mL of 100% ethanol was added. The RNA pellet was then resuspended in RNase-free water. No silica-based membrane and no microspin technology were used for this extraction method (Figure 1).