Preservation of milk in liquid nitrogen during sample collection does not affect the RNA quality for RNA-seq analysis.

BACKGROUND: Standard procedures for milk sample collection for transcriptome analysis use ice as preservation method, which can affect the RNA stability and requires immediate sample processing. These problems would be eased if the milk samples could be snap-frozen in liquid nitrogen. This study describes the applicability of a new method for milk sample collection and subsequent RNA extraction from milk fat globules, determining whether the quality, integrity and quantity of the RNA extracts met the minimum requirements for downstream RNA-seq. RESULTS: The quality of the extracts measured by A(260/280) ratio and the Integrity and Quality (IQ) values obtained fulfilled the reference values of 1.9 - 2.1 (P1 < 0.05 and P2 < 0.05) and ≥ 9 (P < 0.05), respectively. However, the RNA Integrity Number (RIN), based on rRNA 18S and 28S analysis, was 3.59 (P > 0.05) and failed to meet the RIN ≥ 7 benchmark for RNA-seq (P > 0.05). Milk fat globules contain low molecular-weight RNA fragments and minimal 18S and 28S rRNA, suggesting low RIN values were inherent to sample type. Likewise, the RNA concentration from milk fat globules were generally low (120.43 ± 22.27 ng/µL, 102.87 ± 15.64 ng/µL and 109.43 ± 22.69 ng/µL, measured by Nanodrop, Qubit HS and QuanTI Ribogreen, respectively). Nevertheless, RNA-seq yielded 52.7 million paired-end reads per sample. The raw reads passed all quality control parameters having the same sequence-read lengths (151 bp), 100% base-coverage, 49% GC base content, and base quality scores of 36, enabling successful transcriptome profiling. Moreover, milk proteins were identified as the most abundant transcripts in MFG in the analysis of the most expressed genes, indicating that the sequenced reads would accurately reflect the transcriptome of this milk fraction. CONCLUSIONS: Milk preservation in liquid nitrogen is a suitable sample collection method that overcomes the limitations of immediate sample processing required if ice is used. Thus, this procedure, together with the subsequent RNA isolation from milk fat globules and its sequencing by RNA-seq, would provide a practical and a non-invasive method for measuring the mammary epithelial cell transcriptome, improving the feasibility of conducting studies related to mammary gland and lactation physiology.