Characterisation of the miRNA content of human milk: novel molecules with multifunctional significance for the mother and the infant

[Truncated] Human milk (HM) is recognized as the optimal food for term infants, meeting their nutritional, protective and developmental needs especially in the first six months of life. In recent years, a plethora of novel bioactive components have been discovered in HM, with potential regulatory functions in both the mother and the infant during the breastfeeding period. Amongst these are microRNAs (miRNAs), non-coding RNA molecules that are abundant in HM and other mammals’ milks. Particularly in HM, miRNAs and their regulatory roles are still largely unexplored, and the effects of factors that may influence the content and composition of HM in these molecules, such as milk removal or the stage of lactation, are still unknown. Importantly, the origin of miRNAs in HM has not yet been investigated, whether it be the maternal bloodstream and/or the mammary epithelium.

Prior to investigating the miRNA content of HM, extraction of intact total RNAs and miRNAs from each HM fraction (cells, lipids and skim milk) needs to be optimized. In the first experimental chapter of this thesis, eight commercially available miRNA extraction kits were evaluated in each HM fraction, testing different extraction methods (filter column; phenol/guanidine; or a combination of filter column and phenol/guanidine). The efficacy and quality of each kit were tested in 472 samples of different HM fractions obtained from 29 lactating women. The extracted miRNAs were measured using a NanoDrop (for total quantity) and a Bioanalyzer (for small RNA to miRNA ratio). Two individual highly expressed miRNAs in HM were also quantified using qPCR. The column-based phenol-free method was found to be the most effective method for extraction of miRNA from all three HM fractions, with specific kits performing better than others depending on the HM fraction. Although skim milk has been mainly used previously for miRNA studies in HM, here the milk cell fraction yielded higher total RNA and miRNA quantities than the lipid and skim milk fractions, with the lowest quantities found in skim milk. These results suggested that careful consideration of sampling and the miRNA extraction method for each fraction of HM should be taken for HM miRNA studies, such as profiling and functional analyses.