title: MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p.
abstract: OBJECTIVE
  Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours.
  
  
  METHODS
  Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p). Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method.
  
  
  RESULTS
  MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H) allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b) to normalize raw quantification cycle (Cq) data. We identified a daily oscillation of miR-16-5p.
  
  
  CONCLUSIONS
  Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.
authors.affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Floris
authors.affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Billard
authors.affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France.
authors.forename: Clair-Yves
authors.initials: CY
authors.lastname: Boquien
authors.affiliation: University of Nantes, Hospital of Mother and Child, Nantes, France.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Joram-Gauvard
authors.affiliation: University of Nantes, Hospital of Mother and Child, Nantes, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Simon
authors.affiliation: University of Nantes, Hospital of Mother and Child, Nantes, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Legrand
authors.affiliation: University of Nantes, Hospital of Mother and Child, Nantes, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Boscher
authors.affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France; University of Nantes, Hospital of Mother and Child, Nantes, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Rozé
authors.affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France.
authors.forename: Francisco
authors.initials: F
authors.lastname: Bolaños-Jiménez
authors.affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Kaeffer
mesh_terms: D002940:Circadian Rhythm; D005260:Female; D006801:Humans; D007234:Infant, Premature; D035683:MicroRNAs / Q000032:analysis*; D008895:Milk, Human / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
chemical_list: C505362:MIRN16 microRNA, human; D035683:MicroRNAs