Milk fat collection, RNA extraction, and real-time quantitative PCR

Procedures for milk fat collection, RNA extraction, and real-time quantitative PCR were performed according to those described by Nichols et al. [17], with some modifications. During morning milking on d 5 of each period, 10 mL of milk was collected by hand from individual cows within 10 min from the time the milking machine was removed. This approach allowed each sample to be processed individually, thus minimizing the amount of time between alveolar ejection of fresh milk and placing the collected milk fat into RNase inhibitor (TRIzol Reagent; Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Samples were immediately centrifuged at 2000×g for 10 min at room temperature to facilitate separation of the fat fraction. This centrifugation process of whole milk is expected to render the contribution of other cellular material (immune cells, sloughed epithelial cells, stem cells) negligible in the milk fat layer [22, 23]. Approximately 1 g of the supernatant cream layer was transferred into 6 mL TRIzol Reagent, mixed vigorously, snap frozen in liquid N₂, and stored at − 80 °C until RNA extraction. Total RNA was isolated from milk fat according to TRIzol manufacturer’s instructions for handling samples with a high fat content. Total RNA concentrations and purity were determined by optical density measurement using a Nano-Drop ND-1000 (ThermoFisher Scientific). The average 260/280 absorbance ratio of total RNA samples was adequate at 1.89 ± 0.117 (mean ± SD) and the average RNA concentration was 115 ± 64.1 ng/μL (mean ± SD). The mean RNA integrity number (RIN) was 4.5 ± 2.30 (mean ± SD). The low concentration of RNA found with this procedure may have impacted RIN measurement [24]. This RIN is low by conventional standards [25], but was consistent across all samples, suggesting it was inherent to the sample type and not due to degradation of only a few samples. An aliquot of 100 ng total RNA was reverse transcribed in a total volume of 20 μL with Superscript III (100 U; ThermoFisher Scientific) in the presence of random hexamers (250 ng; Roche, Almere, the Netherlands), dithiothreitol (0.5 mmol/L; ThermoFisher Scientific) and dNTPs (0.5 mmol/L; Roche) at 25 °C for 5 min and 50 °C for 1 h. After inactivation of the enzyme (70 °C, 15 min), cDNA was stored at − 80 °C until further analysis.

Specific primers (Eurogentec, Maastricht, the Netherlands) were intron-spanning and designed to yield amplicons in the range of 100 and 180 bp (Table ​(Table1)1) with efficiencies of 90–100%. Templates were amplified after a preincubation of 10 min at 95 °C, followed by amplification for 40 cycles (10 s at 95 °C, 5 s at 60 °C, and 5 s at 72 °C) on a 7500 Fast Real-Time PCR System (Applied Biosystems Deutschland GmbH, Darmstadt, Germany) by using the SensiMix SYBR Low-ROX kit (Bioline UK Ltd., London, UK). All melting curves confirmed that a single amplicon was produced. As a control, a standard cDNA was included on each qPCR plate, resulting in a single and similar amplicon for the standard and cDNA samples as evidenced by melting curve analysis. As internal standards, expression of housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein S9 (RPS9), and ubiquitously expressed transcript isoform 2 (UXT) were analysed. NormFinder [26] identified RPS9 as the most stable gene across experimental period and treatment. Fold changes in gene expression relative to LAA-C were calculated by the 2^−ΔΔCt method [27] using RPS9 expression as the reference gene.

Primer sequences used for real-time quantitative PCR

¹ACC1 = acetyl-CoA carboxylase 1; CASP3 = caspase 3; CCND1 = cyclin D1; CDC42BPA = CDC42 binding protein kinase alpha; CSN2 = β-casein; DDIT3 = DNA damage-inducible transcript 3; DDIT4 = DNA damage inducible transcript 4; EIF2A = eukaryotic initiation factor 2, α subunit; EIF2AK3 = eukaryotic translation initiation factor 2, alpha kinase 3; EIF2B5 = eukaryotic translation initiation factor 2B, ε subunit; EIF4E = eukaryotic translation initiation factor 4E; FASN = fatty acid synthase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HSPA5 = H3 histone, family 3A; IDH1 = isocitrate dehydrogenase 1, cytosolic; LALBA = α-lactalbumin; ME2 = malic enzyme 2, mitochondrial; MYC = MYC proto-oncogene; PCK2 = phosphoenolpyruvate carboxykinase 2, mitochondrial; PPP1R15A = protein kinase AMP-activated catalytic, α1 subunit; PRKAA1 = protein kinase AMP-activated catalytic, α1 subunit; RPL15 = ribosomal protein L15; RPS6 = ribosomal protein S6; RPS6KB1 = ribosomal protein S6 kinase B1; RPS9 = ribosomal protein S9; UXT = ubiquitously expressed prefoldin like chaperone; XBP1s = X-box binding protein 1, spliced; XBP1u = X-box binding protein 1, unspliced