Immune-related miRNA levels in human breast milk (n = 5) and serum (n = 6) of healthy subjects measured by qRT-PCR.

Comparison by qRT-PCR of immune-related miRNAs in the breast milk obtained before 6 months of age (n = 5) (before the infants received complementary food) and from 6 to 12 months of age (n = 13) (when infants were receiving complementary food).

Exosome-like vesicles from human breast milk display the typical size (30--300 nm) and ultrastructure of electron-dense and electron-lucent microvesicles (arrows). miRNAs in the vesicles within breast milk are received by infants and facilitate many aspects of the infant's development. Scale bar, 100 nm.

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.

Expression profile of miRNAs in human breast milk. (a) RNA isolation from breast milk. RNA from breast milk was detected using a Bioanalyzer. The milk contains a substantial amount of small RNA (<300 nt), but very little or no ribosomal RNA (18S and 28S rRNA). (b--d) Expression profiling of miRNAs in human breast milk from miRNA microarray. (b) miRNAs in breast milk that were detected or hardly detected by microarray analysis. (c) Upper panel: a clustering analysis of the expression of breast milk miRNAs before and after the infants received complementary food. Samples 1, 2 and 3 were from different mothers. Breast milk samples were taken during the first 6 months after birth (0--6) and during the subsequent months (6+). Lower panel: Strongly expressed miRNA in breast milk before and after the infants received complementary food. (d) Correlation of gene expression signals between samples obtained from the same mother during the first 6 months and during the following 6 months are shown. The relationship between samples from different mothers was not as close.