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Washing the milk fat globule minimizes cellular contamination without compromising mRNA quality

Milk fat globules (MFG) are a rich source of lactocyte mRNA. We examined if the lipid phase of centrifuged whole milk contains extraneous cells (presence of nucleic matter); if washing the MFG will reduce cell contamination; and factors associated with RNA quality. Early (E, day 2) and Mature (M, day 24–424) fresh human milk samples were centrifuged x 10 min to obtain MFG using 3 methods: Soft (300 g), Hard (15,000 g) and Washed (hard spin, re‐suspend MFG in buffer, repeat x 2). MFG (N=24 E and 31 M) were stored at −80 in TRIzol, RNA isolated, and assayed for RNA integrity number (RIN). The transcriptome was sequenced (1 Washed E and 6 Washed M to date; Illumina HiSeq2000). DAPI staining of the MFG confirmed cell presence in unwashed E and M. Washing dramatically reduced cells in E, and eliminated cells in M; Sequenced Washed E and M had no expression of CD45 (leukocyte marker) and Western blot revealed heavy bands of â‐Actin and no visible Lamin. RIN (N=55, Median=8.1) was not correlated with processing method, day postpartum, days in TRIzol, or days stored as isolated RNA. Among E samples (N=24), RIN was correlated with minutes from collection to TRIzol (range, 50–155; r=−0.48) and hours from collection until stage II lactogenesis (range, −14 to 44; r=−0.35). Overall, 31% were unsuitable for sequencing (RIN < 7.0), likely due to RNAase contact during sample collection. Funded by Cincinnati Diabetes and Obesity Center and HD1302.