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MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p.

Floris I, Billard H, Boquien CY, Joram-Gauvard E, Simon L, Legrand A, Boscher C, Rozé JC, Bolaños-Jiménez F, Kaeffer B - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France.

ABSTRACT

Background and aims: 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.

Materials and 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.

Perspectives: Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.

No MeSH data available.


Related in: MedlinePlus

MiRNA levels measured in lipids and skim milk fractions at different lactation periods.Cq values by box-and-whisker plots (1–99 percentile) of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) measured A) in lipids and B) in skim milk.
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pone.0140488.g002: MiRNA levels measured in lipids and skim milk fractions at different lactation periods.Cq values by box-and-whisker plots (1–99 percentile) of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) measured A) in lipids and B) in skim milk.

Mentions: The same milk samples previously mentioned (Table 1) were purified for their lipids and skim milk fractions, as described in the materials and methods. Lipids and skim milk fractions respectively gave RNA concentrations ranging between 84.9–208.0 ng/μL (mean = 148 ng/μL, SD = 47.8 ng/μL, n = 15) and 103.1–264.0 ng/μL (mean = 161 ng/μL, SD = 41.4 ng/μL, n = 15). In addition, we tested the stability of the five miRNA controls and miR-146b within month 2 of lactation in lipids and skim milk. Raw Cq data of each miRNA were represented in a box-and-whisker plots (1–99 percentile) for both fractions (Fig 2A and 2B). Surprisingly, miR-146b was the most stable in lipids (mean Cq = 26.57, SD = 0.52) and in skim milk (mean Cq = 25.47, SD = 0.71). In skim milk, let-7d (mean Cq = 31.35, SD = 1.0) and miR-21 (mean Cq = 28.15, SD = 1.0) were also stable, while miR-16, let-7g and let-7a showed higher variability (Cq SD > 1.8). In lipids, let-7d (mean Cq = 34.37, SD = 0.92), let-7a (mean Cq = 30.69m SD = 1.0) and miR-21 (mean Cq = 29.9, SD = 1.26) were also stable, whilst miR-16 and let-7g showed higher variability (Cq SD > 1.4).


MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p.

Floris I, Billard H, Boquien CY, Joram-Gauvard E, Simon L, Legrand A, Boscher C, Rozé JC, Bolaños-Jiménez F, Kaeffer B - PLoS ONE (2015)

MiRNA levels measured in lipids and skim milk fractions at different lactation periods.Cq values by box-and-whisker plots (1–99 percentile) of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) measured A) in lipids and B) in skim milk.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608744&req=5

pone.0140488.g002: MiRNA levels measured in lipids and skim milk fractions at different lactation periods.Cq values by box-and-whisker plots (1–99 percentile) of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) measured A) in lipids and B) in skim milk.
Mentions: The same milk samples previously mentioned (Table 1) were purified for their lipids and skim milk fractions, as described in the materials and methods. Lipids and skim milk fractions respectively gave RNA concentrations ranging between 84.9–208.0 ng/μL (mean = 148 ng/μL, SD = 47.8 ng/μL, n = 15) and 103.1–264.0 ng/μL (mean = 161 ng/μL, SD = 41.4 ng/μL, n = 15). In addition, we tested the stability of the five miRNA controls and miR-146b within month 2 of lactation in lipids and skim milk. Raw Cq data of each miRNA were represented in a box-and-whisker plots (1–99 percentile) for both fractions (Fig 2A and 2B). Surprisingly, miR-146b was the most stable in lipids (mean Cq = 26.57, SD = 0.52) and in skim milk (mean Cq = 25.47, SD = 0.71). In skim milk, let-7d (mean Cq = 31.35, SD = 1.0) and miR-21 (mean Cq = 28.15, SD = 1.0) were also stable, while miR-16, let-7g and let-7a showed higher variability (Cq SD > 1.8). In lipids, let-7d (mean Cq = 34.37, SD = 0.92), let-7a (mean Cq = 30.69m SD = 1.0) and miR-21 (mean Cq = 29.9, SD = 1.26) were also stable, whilst miR-16 and let-7g showed higher variability (Cq SD > 1.4).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: UMR-1280, INRA, University of Nantes, Physiologie des Adaptations Nutritionnelles, Nantes, France.

ABSTRACT

Background and aims: 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.

Materials and 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.

Perspectives: Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.

No MeSH data available.


Related in: MedlinePlus