Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation.
MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column-based phenol-free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area.
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia.|College of Applied Medical Sciences, Majmaah University, Almajmaah, Riyadh, Saudi Arabia.
Mohammed
M
Alsaweed
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia.
Anna R
AR
Hepworth
Centre for Biotechnology and Interdisciplinary Sciences, Deakin University, Victoria, Australia.
Christophe
C
Lefèvre
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia.
Peter E
PE
Hartmann
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia.
Donna T
DT
Geddes
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia.
Foteini
F
Hassiotou
D001940:Breast / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007223:Infant; D007774:Lactation / Q000378:metabolism*; D050356:Lipid Metabolism / Q000235:genetics; D035683:MicroRNAs / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008895:Milk, Human / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*
D035683:MicroRNAs; D012313:RNA
BREASTMILK; CELLS; FAT; FAT GLOBULE; HUMAN MILK; LIPIDS; MILK FRACTIONS; RNA; SKIM MILK; microRNA