Next generation sequencing of the washed milk fat globule transcriptome

Abstract

Milk fat globules (MFG) contain cytoplasmic crescents, which are a rich source of lactocyte mRNA. Our aim was to characterize the transcriptome of washed human MFG using Next Generation Sequencing (NGS). Fresh milk was collected from 7 predominately breastfeeding women (2–172 d postpartum) and centrifuged at 15,000g for 10 minutes. The lipid layer was washed twice in PBS to purify the MFG. Poly(A)RNA was extracted and sequenced (Illumina HiSeq2000), averaging 15.3 million 50bp single reads per sample; 27,294 unique transcript variants were expressed, comprising 16,924 unique genes. Blind clustering analysis revealed a distinct transcriptional profile for the participant recovering from mastitis. Functional analyses revealed key genes expressed in the lactocyte that are not yet functionally annotated. The unprecedented dynamic range of NGS enabled us to determine that the top 5 abundant milk protein genes contribute 40% of the mRNA, suggesting that minor abundance transcripts may not be actively down‐regulated by altered transcription factor binding or chromatin modification, but instead by dilution, a mechanism by which lactocytes could dramatically alter phenotype by active up‐regulation of a few key genes. As the first NGS of the human MFG, these data will inform future applications of this powerful tool in lactation research. Funded by Cincinnati Diabetes and Obesity Center (LAN‐R) and NICHD (ALM).