Optimizing Protocols for MicroRNA Profiling of Infant and Toddler Stool.

Background: MicroRNAs (miRNAs) are increasingly being investigated as potential biomarkers for child development and disease. Although a growing number of studies are utilizing infant and toddler stool for transcriptomic analyses, no studies have compared protocols for preserving and extracting miRNAs from this specimen type, despite unique challenges, including abundant levels of RNAses and microbial RNA. Methods: To address this, we first compared three commercially available kits and four preservation methods for their ability to yield high quality RNA from infant and toddler stool (Phase 1). RNA quality was determined by fragment analyzer. Results: Of the three RNA extraction kits compared, Zymo BIOMICs yielded the highest overall RNA Quality Number (RQN) (median (range) RQN 9.4 (5.7-10.0)). Of the four preservation methods tested, stool collected in RNAlater and Zymo DNA/RNA Shield Fecal 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 significantly from each other ( Conclusions: Collecting infant and toddler stool in either RNAlater or Zymo DNA/RNA Shield Fecal Collection Tubes, when paired with RNA extraction using the Zymo BIOMICs extraction kit, yielded high-quality RNA with similar human miRNA profiles. Moreover, of the 42 miRNAs that were detected, several (i.e., miR-194a-3p, miR-200c-3p, miR-26a-5p) are thought to contribute to overall gut homeostasis. These findings may inform protocols for future studies that aim to profile miRNAs in infant and toddler stool to evaluate their potential utility as biomarkers for children's health.