Jiménez-Montenegro Lucía, Alfonso Leopoldo, Soret Beatriz, Mendizabal José A, Urrutia Olaia
IS-FOOD, School of Agricultural Engineering and Biosciences, Public University of Navarre (UPNA), Campus de Arrosadia, Pamplona, 31006, Spain.
BMC Genomics. 2025 May 24;26(1):525. doi: 10.1186/s12864-025-11707-6.
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.
The quality of the extracts measured by A 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.
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.
用于转录组分析的牛奶样本采集标准程序采用冰作为保存方法，这会影响RNA稳定性且需要立即进行样本处理。如果牛奶样本能够在液氮中速冻，这些问题将得到缓解。本研究描述了一种新的牛奶样本采集方法以及随后从乳脂肪球中提取RNA的适用性，确定RNA提取物的质量、完整性和数量是否满足下游RNA测序的最低要求。
通过A比率测量的提取物质量以及获得的完整性和质量（IQ）值分别满足1.9 - 2.1（P1 < 0.05且P2 < 0.05）和≥9（P < 0.05）的参考值。然而，基于rRNA 18S和28S分析的RNA完整性数值（RIN）为3.59（P > 0.05），未达到RNA测序的RIN≥7基准（P > 0.05）。乳脂肪球含有低分子量RNA片段以及极少的18S和28S rRNA，这表明低RIN值是样本类型所固有的。同样，来自乳脂肪球的RNA浓度普遍较低（分别通过Nanodrop、Qubit HS和QuanTI Ribogreen测量，为120.43±22.27 ng/µL、102.87±15.64 ng/µL和109.43±22.69 ng/µL）。尽管如此，RNA测序每个样本产生了5270万个双端读数。原始读数通过了所有质量控制参数，具有相同的序列读长（151 bp）、100%碱基覆盖率、49%的GC碱基含量以及36的碱基质量得分，从而能够成功进行转录组分析。此外，在分析表达量最高的基因时，牛奶蛋白被确定为乳脂肪球中最丰富的转录本，这表明测序读数能够准确反映该牛奶组分的转录组。
在液氮中保存牛奶是一种合适的样本采集方法，克服了使用冰时需要立即进行样本处理的局限性。因此，该程序连同随后从乳脂肪球中分离RNA并通过RNA测序进行测序，将为测量乳腺上皮细胞转录组提供一种实用且非侵入性的方法，提高开展与乳腺和泌乳生理学相关研究的可行性。
