Preserving RNA quality when freezing of human milk samples must occur before extraction: Validation of methods utilized in a multi-center cohort study

Recent multicenter studies aim to define the effects of mothers’ blood glucose levels during pregnancy on mammary gland function and breast milk composition. Gene expression measured in cells in milk can serve as a liquid biopsy to evaluate the molecular biology of the mammary gland. Critical to this aim is reproducible and high-quality extraction of RNA from milk and harmonized collection protocols from across centers. To address this, we performed a study to optimize milk RNA quality metrics where samples are collected at multiple centers and shipped to a central laboratory for processing and analysis. Lactating mothers provided breast milk following informed consent. The treatments of the samples were as follows: (1) 200 µL of fresh, never frozen milk used as control (FRESH); (2) 1.7 or 5 mL of milk frozen and thawed on ice before adding TRIzol (FRZ); (3) 200 µL of milk frozen and thawed after adding TRIzol (FRZ 200); and (4) 200 µL of milk with 20 µL of RNA preservative added, frozen and thawed after adding TRIzol (FRZ + INH). In all scenarios, RNA was extracted using TRIzol followed by purification with a Qiagen RNeasy Mini kit. For the FRZ 200 and FRZ 200 + INH samples, TRIzol was directly added at the start of thawing, before extraction. Outcomes included RNA concentration, RNA purity (260/280 ratio), RNA fragmentation (DV 200), RNA integrity number (RIN), quantification via Qubit fluorescence-based assays, and visualization of RNA size on an Agilent TapeStation. A RIN cut-off value ≥ 7 indicated acceptable quality for transcriptomics studies. RNA quality metrics were modeled as continuous outcomes using linear mixed-effects regression models. For FRESH milk ( n = 15) the estimated marginal mean (EMM) RIN was 7.48 (SE 0.29). For FRZ 200, ( n = 22), the EMM RIN was 6.94 (SE 0.26). Samples of FRZ 200 + INH ( n = 21) had an EMM RIN of 7.81 (SE 0.26). However, FRZ ( n = 19) samples demonstrated markedly reduced RNA integrity with an EMM RIN of 1.92 (SE 0.26). RIN was not significantly different in FRESH versus FRZ 200 or FRZ 200 + INH milk. FRZ 200 + INH samples showed a statistically significant improvement in RIN compared to FRZ 200 ( p = 0.0038). RNA quantities were sufficient for sequencing across all treatments. The addition of TRIzol directly to a 200 µL aliquot of milk at the start of thawing provided the highest integrity of extracted milk RNA, as measured by RIN. Adding RNase inhibitor at the time of sample collection, prior to freezing, also enhanced RNA integrity. We have developed a method to optimize the integrity of RNA from frozen human milk samples. This is a crucial methods improvement for multi-center studies where freezing of milk samples is often required prior to RNA extraction and analysis. These results can inform reproducible research protocols for evaluating the use of breastmilk as a liquid biopsy for mammary gland function.