General characterization of miRNAs in breast milk. (a--c) The stability of breast milk miRNAs. Total RNA was extracted and then analyzed by qRT-PCR. Breast milk was (a) incubated with RNase A/T for 3 h at 37°C, (b) subjected to three freeze-thaw cycles or (c) treated for 3 h in a low pH solution (pH 1) before RNA extraction. Representative data are shown. (d) Expression of miR-181a and miR-17 derived from CD63-positive exosomes isolated from human breast milk (0.3 ml) from different mothers (n = 4). Human breast milk was immunoprecipitated with anti-CD63 antibody or isotype control.