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An automated method to quantify milk fat globules and sources of RNA in milk
Author(s) -
Hartono Stella R,
Ventimiglia Frank,
Smilowitz Jennifer T,
German J Bruce,
Korf Ian,
Lemay Danielle G
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.624.4
Subject(s) - globules of fat , acridine orange , rna , lactation , biology , food science , cytoplasm , milk fat , chemistry , chromatography , microbiology and biotechnology , gene , biochemistry , genetics , apoptosis , linseed oil , pregnancy
Given the recent use of milk RNA to assay the mammary gland transcriptome, there is a need to visualize and quantify the sources of milk RNA in an unbiased, accurate, efficient manner. We developed a method to quantify globules and RNA sources from whole milk stained with acridine orange (AO). Three channels—a differential interference contrast image, a fluorescence channel to capture AO‐RNA, and a second fluorescence channel to capture AO‐DNA—were captured from slides using an Olympus VS110 whole slide scanner. Software was developed to identify sizes and locations of globules, cytoplasmic crescents, and nucleated cells and to compute summary statistics. A 300MB image containing 20,000 globules can be processed in under a minute and multiple images can be processed in batch. The method's accuracy was checked by manually comparing processed and original images. Whole milk samples from three human subjects were tested. Crescents were associated with 6.92%, 8.06%, and 6.76% of milk fat globules, respectively; 14.1%, 14.2% and 0.8% of the whole milk RNA was estimated to be of nucleated cell origin. When milk from the first subject (14.1% cells) was centrifuged into fat and skim fractions, the percentage of RNA attributable to nucleated cells in the milk fat fraction dropped to 8.7%. This method of quantification is expected to be generally applicable to other lactation stages and species. Funded by NIH P51RR000169.