Methods for Preserving Human Milk Cellular and Milk Fat Globule RNA

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 RNA preservation methods for milk cells and MFG, using RNAlater for stabilization before freezing. Human milk samples (n = 26) were collected from lactating participants and either frozen (standard practice) or mixed with RNAlater (1:1, v/v) before freezing. RNA was extracted from separated cellular and MFG fractions and assessed for concentration, quality (RNA quality number-RQN and 28 S/18S ratio), and gene expression (ACTB, LALBA, PRLR, PTPRC) using RT-qPCR. Samples preserved with RNAlater showed significantly improved RNA integrity, particularly in the MFG fraction, compared to those frozen without RNAlater. Gene expression was largely stable across preservation methods. Delays in mixing with RNAlater led to declining RQN values in milk cell fractions, underscoring the need for prompt stabilization. Lastly, we show that this method can be used in low-resource countries by extracting RNA from samples collected in a randomized clinical trial in Guinea-Bissau and shipped to the US for analysis. This procedure led to improved yield and integrity of these samples. These findings demonstrate that RNAlater pre-freezing stabilization enhances RNA quality and yield, and supports its use for milk gene expression analysis. This approach provides a practical, scalable solution for RNA preservation in clinical and field research, including remote and low-resource settings, as it requires minimal experience and equipment.