Peer-reviewed article
Optimizing protocols for microRNA profiling of infant and toddler stool.
David A Armstrong, Shannon M Soucy, Meghan E Muse, Fred W Kolling, Heidi W Trask et al. ·
Summary
Written by AI, not yet AICS's view · claude-haiku-4-5-20251001This is a technical methods paper comparing laboratory protocols for extracting microRNA from stool samples of infants and toddlers under 2 years old. The study tested different commercial extraction kits and preservation methods to identify the best approach for maintaining RNA quality. The research found that two specific preservation methods and one extraction kit produced high-quality results suitable for microRNA analysis.
Key points
- Zymo BIOMICs extraction kit yielded the highest RNA quality (median RQN 9.4)
- RNAlater and Zymo DNA/RNA Shield Faecal Collection Tubes produced similar high-quality results (median RQN 9.8 and 9.4 respectively)
- miRNA profiles from samples preserved in the two methods did not differ significantly
- 42 human miRNAs were successfully detected and measured across test samples
Limitations
This is a technical protocol optimization study with very small sample sizes (n=4 infant donors) focused on laboratory methodology rather than child health outcomes or well-being. It does not evaluate any health intervention or measure any impact on children's health or development.
Abstract
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 <2 years of age (infant/toddler). 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 <2 years of age in either RNAlater or Zymo DNA/RNA Shield Faecal Collection Tubes, when paired with RNA extraction using the Zymo BIOMICs extraction kit, yielded high-quality RNA with similar human miRNA profiles.