Characterizing human milk RNA degradation over time to optimize storage of human milk for RNA quality

Authors: CRIMMINS, MEGHAN - University Of Minnesota; REED, BRITTANY - Arkansas Children'S Nutrition Research Center (ACNC); DA CRUZ, LARISSA - University Arkansas For Medical Sciences (UAMS); SIMS, CLARK - University Arkansas For Medical Sciences (UAMS); SOBIK, SARAH - University Arkansas For Medical Sciences (UAMS); BORSHEIM, ELISABET - University Arkansas For Medical Sciences (UAMS); PORTER, CRAIG - University Arkansas For Medical Sciences (UAMS); SIMMEN, ROSALIA - University Arkansas For Medical Sciences (UAMS); ANDRES, ALINE - University Arkansas For Medical Sciences (UAMS)

Submitted to: Journal of Mammary Gland Biology and Neoplasia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/16/2026
Publication Date: 1/28/2026
Citation: Crimmins, M.R., Reed, B., Da Cruz, L.L., Sims, C.R., Sobik, S., Borsheim, E., Porter, C., Simmen, R.C., Andres, A. 2026. Characterizing human milk RNA degradation over time to optimize storage of human milk for RNA quality. Journal of Mammary Gland Biology and Neoplasia. 31:Article 5(2026). https://doi.org/10.1007/s10911-026-09597-9.
DOI: https://doi.org/10.1007/s10911-026-09597-9

Interpretive Summary: Human milk contains bioactive RNA molecules that may contribute to infant health and development, but little information exists on how storage conditions affect RNA stability, which can compromise research quality and comparability across studies. To address this gap, researchers conducted a clinical study with 30 lactating women in central Arkansas to evaluate how temperature, storage duration, and use of a preservation solution influence RNA quality in milk samples stored for up to six months. Results showed that RNA quality declined over time without preservation, while adding RNAlater helped maintain mRNA integrity during frozen storage, particularly at -80 °C. In contrast, miRNAs remained stable when samples were frozen without preservatives, and RNAlater did not improve their stability. These findings provide practical guidance for human milk sample handling and storage, helping researchers preserve RNA quality, improve data reliability, and strengthen future studies aimed at understanding the biological role of human milk in supporting infant growth and development.

Technical Abstract: Human milk is rich in bioactive components beyond nutrition, including RNAs—especially miRNAs. It is the most RNArich biofluid. While storage effects on other milk components are well studied, RNA preservation has been minimally explored, primarily focusing on miRNAs in exosomes under refrigeration. The goal of this clinical study was to determine optimal collection and storage methods to preserve RNA quantity and quality in human milk, predominantly focusing on RNA stability in research laboratory settings when isolation of milk fat globules is not feasible. The study recruited 30 lactating women in central Arkansas. Milk samples were collected in-clinic and processed immediately for baseline RNA data. Remaining aliquots were stored at 4 °C, -20 °C, or -80 °C with or without RNAlater. RNA was extracted at 4 h, 24 h, 1 week, 4 weeks, 12 weeks, and 24 weeks using Qiagen miRNeasy and MinElute kits. RNA quality (RINe) and concentration (ng/µL) were assessed with Agilent TapeStation with additional quality analysis through qPCR. RNA quality declined over time at all storage temperatures in samples without RNAlater. Addition of RNAlater prevented decreases in RINe scores at most time points, maintaining RINe>7 for up to 6 months, though it reduced total RNA concentration. qPCR also showed improved mRNA stability with RNAlater. miRNAs were analyzed using Agilent Bioanalyzer Small RNA kits with qPCR for quality analysis. RNAlater was not beneficial for miRNA preservation, and miRNAs remained stable when stored frozen without RNAlater. qPCR verified miRNA stability when frozen. In conclusion, RNA in human milk degrades over time. For mRNA analysis, storage with RNAlater at -80 °C is recommended. For miRNA analysis, storage at -20 °C or -80 °C without preservatives is optimal. These findings offer preliminary guidance for HM RNA storage, and researchers are advised to conduct method testing before full study implementation.