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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...

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Autores principales: Alsaweed, Mohammed, Hepworth, Anna R., Lefèvre, Christophe, Hartmann, Peter E., Geddes, Donna T., Hassiotou, Foteini
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042114/
https://www.ncbi.nlm.nih.gov/pubmed/25925799
http://dx.doi.org/10.1002/jcb.25207
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author Alsaweed, Mohammed
Hepworth, Anna R.
Lefèvre, Christophe
Hartmann, Peter E.
Geddes, Donna T.
Hassiotou, Foteini
author_facet Alsaweed, Mohammed
Hepworth, Anna R.
Lefèvre, Christophe
Hartmann, Peter E.
Geddes, Donna T.
Hassiotou, Foteini
author_sort Alsaweed, Mohammed
collection PubMed
description 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. J. Cell. Biochem. 116: 2397–2407, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.
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spelling pubmed-50421142016-10-03 Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation Alsaweed, Mohammed Hepworth, Anna R. Lefèvre, Christophe Hartmann, Peter E. Geddes, Donna T. Hassiotou, Foteini J Cell Biochem Articles 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. J. Cell. Biochem. 116: 2397–2407, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. John Wiley and Sons Inc. 2015-08-14 2015-10 /pmc/articles/PMC5042114/ /pubmed/25925799 http://dx.doi.org/10.1002/jcb.25207 Text en © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs (http://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Articles
Alsaweed, Mohammed
Hepworth, Anna R.
Lefèvre, Christophe
Hartmann, Peter E.
Geddes, Donna T.
Hassiotou, Foteini
Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation
title Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation
title_full Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation
title_fullStr Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation
title_full_unstemmed Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation
title_short Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation
title_sort human milk microrna and total rna differ depending on milk fractionation
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042114/
https://www.ncbi.nlm.nih.gov/pubmed/25925799
http://dx.doi.org/10.1002/jcb.25207
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