Characterizing Human Milk RNA Degradation Over Time to Optimize Storage of Human Milk for RNA Quality

Human milk is rich in bioactive components beyond nutrition, including RNAs-especially miRNAs. It is the most RNA-rich 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.