Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs.
By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems.
Hata
Taketoshi
T
Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507, Japan.
Murakami
Kosuke
K
Nakatani
Hajime
H
Yamamoto
Yasunari
Y
Matsuda
Tsukasa
T
Aoki
Naohito
N
D000818:Animals; D002417:Cattle; D055354:Exosomes / Q000737:chemistry; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification*; D008892:Milk / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D022161:Transport Vesicles / Q000737:chemistry* / Q000502:physiology; D014461:Ultracentrifugation
D035683:MicroRNAs; D012333:RNA, Messenger