Methods for Preserving Cellular and Milk Fat Globules RNA from Human Milk Samples

Human breast milk contains RNA in various fractions, including milk cells and milk fat globules (MFG), making it a valuable resource for studying lactation physiology. However, preserving RNA integrity, especially in low-resource or at-home collection settings, is challenging due to rapid RNA degradation. This study aimed to evaluate and optimize RNA preservation methods for BM cells and MFGs, including a simplified protocol using RNAlater for stabilization prior to freezing. Human milk samples (n = 16) were collected from lactating participants and either frozen directly (standard practice) or mixed with RNAlater (1:1, v/v) before freezing. RNA was extracted from separated milk cell and MFG fractions and assessed for concentration, quality (RNA quality number-RQN and 28S/18S ratio), and gene expression (ACTB, LALBA, PRLR, PTPRC) using qPCR. MFG fractions consistently yielded higher RNA concentrations than milk cells. Samples preserved with RNAlater showed significantly improved RNA quality, particularly in the MFG fraction, compared to those frozen without RNAlater. Gene expression was largely stable across preservation methods, though the immune marker gene PTPRC was reduced in RNAlater-treated samples, suggesting shifts in immune cell RNA content. Delays in RNAlater addition led to declining RQN values in milk cell fractions, underscoring the need for prompt stabilization. These findings demonstrate that RNAlater pre-freezing stabilization enhances RNA quality and yield, especially from MFGs, and supports its use for lactocytes gene expression analysis. This approach provides a practical, scalable solution for RNA preservation in clinical and field research, including remote and low-resource settings.