Beckman j 6b

Milk samples were collected from the morning milking on the final day of each period and subjected to RNA analysis using the methods of Brenaut et al. (2012) (link) and Cánovas et al. (2014) (link) with the following modifications. Milk samples (45 mL) were collected into RNase-free sample bottles, transferred into sterile 50 mL conical tubes, and stored on ice. Samples were centrifuged at 2,000 × g for 10 min at 4°C (Beckman J-6B; Beckman Coulter, Inc., Indianapolis, IN) to separate fat from other milk components. The resultant fat was suspended in RNase-free water (1:1), and an equal volume of TRIzol LS (Life Technologies, Grand Island, NY) was added and thoroughly mixed. All samples were stored at -80°C until analysis. Chloroform was added to the thawed samples, they were centrifuged at 4°C and 12,000 × g for 15 min, and the resultant aqueous phase was combined with an equal volume of acid phenol: chloroform-isoamyl alcohol (Ambion, Life Technologies). Samples were centrifuged at 12,000 × g at 4°C for 10 min, and the aqueous phase was combined with an equal volume of 70% ethanol, vortexed, and purified using the RNeasy Mini Kit and treated with DNase I (Qiagen Inc.). A 2 μg aliquot of the resulting RNA was reverse-transcribed using an Omniscript reverse transcriptase kit (Qiagen Inc.), oligo-dT (0.05 μg/μL; Qiagen Inc.), and random decamers (10 μM; Ambion).