Optimizing protocols for microRNA profiling of infant and toddler stool

Despite growing interest in profiling microRNAs (miRNAs) in infant and toddler stool, no studies have compared protocols for preserving and extracting miRNAs from this specimen type. Three commercially available kits and four preservation methods were compared for their ability to yield high quality RNA from children Of the three RNA extraction kits compared, Zymo BIOMICs yielded the highest RNA Quality Number (RQN) (median (range) RQN 9.4 (5.7–10.0)). Of the four preservation methods tested, RNAlater and Zymo DNA/RNA Shield Faecal Collection Tubes yielded the highest two RQNs (median (range) RQN 9.8 (5.7–10.0) and 9.4 (5.4–10.0), respectively), which did not differ from each other (p = 0.47). Subsequently, miRNA-seq was used to compare miRNA profiles for RNA extracted using the Zymo BIOMICs kit from paired aliquots of the same stool sample (n = 4 infant donors) collected into RNAlater and Zymo DNA/RNA Shield Faecal Collection Tubes. The percentage of reads classified as human and the percentage of human reads aligning to miRBase did not differ for samples collected in RNAlater versus Zymo Shield (p = 0.12 and p = 0.86, respectively). Furthermore, after multiple testing correction, normalized miRNA counts did not differ between the two preservatives for any of the 42 human miRNAs detected across the eight samples (pFDR ≥ 0.05). Collecting stool from infants and toddlers