TY - BOOK
T1 - Characterisation of the miRNA content of human milk: novel molecules with multifunctional significance for the mother and the infant
AU - Alsaweed, Mohammed Abdullah I
PY - 2015/11
Y1 - 2015/11
N2 - [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.
AB - [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.
KW - Human milk
KW - MicroRNA
KW - Gene regulation
KW - Development
KW - Lactation
KW - Mammary gland
KW - Infant
KW - Breastfeeding
M3 - Doctoral Thesis
ER -