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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

Differences in miRNA expression levels between whole milk, lipids and skim milk.Raw Cq data of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) analyzed in full milk, lipids, and skim milk, were represented in a Tukey box plot. MiR-21, let-7a and let-7d expression was significantly lower in lipids than in skim and full milk, while no differences were found between skim milk and whole milk. MiR-16, let-7g and miR-146b expression was significantly higher in skim milk fractions than in lipids and in full milk (*p<0.05; **p<0.01; ***p<0.001).
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pone.0140488.g003: Differences in miRNA expression levels between whole milk, lipids and skim milk.Raw Cq data of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) analyzed in full milk, lipids, and skim milk, were represented in a Tukey box plot. MiR-21, let-7a and let-7d expression was significantly lower in lipids than in skim and full milk, while no differences were found between skim milk and whole milk. MiR-16, let-7g and miR-146b expression was significantly higher in skim milk fractions than in lipids and in full milk (*p<0.05; **p<0.01; ***p<0.001).

Mentions: We checked for differences in miRNA distribution between whole milk, lipids and skim milk. In Fig 3, we represented in a graph the mean Cq with SEM of the six measured miRNAs in whole milk or its fractions. MiR-21, let-7d and let-7a Cq values were significantly lower in lipids than in skim milk (respectively p<0.001, p<0.001 and p<0.01) and than in full milk (respectively p<0.001, p<0.001 and p<0.01), whilst no differences were found between skim milk and whole milk. On the other hand, miR-16, let-7g and miR-146b Cq values were significantly higher in skim milk fractions than in lipids (respectively p<0.001, p<0.01 and p<0.01) and whole milk (respectively p<0.001, p<0.05 and p<0.001).


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)

Differences in miRNA expression levels between whole milk, lipids and skim milk.Raw Cq data of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) analyzed in full milk, lipids, and skim milk, were represented in a Tukey box plot. MiR-21, let-7a and let-7d expression was significantly lower in lipids than in skim and full milk, while no differences were found between skim milk and whole milk. MiR-16, let-7g and miR-146b expression was significantly higher in skim milk fractions than in lipids and in full milk (*p<0.05; **p<0.01; ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140488.g003: Differences in miRNA expression levels between whole milk, lipids and skim milk.Raw Cq data of endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) analyzed in full milk, lipids, and skim milk, were represented in a Tukey box plot. MiR-21, let-7a and let-7d expression was significantly lower in lipids than in skim and full milk, while no differences were found between skim milk and whole milk. MiR-16, let-7g and miR-146b expression was significantly higher in skim milk fractions than in lipids and in full milk (*p<0.05; **p<0.01; ***p<0.001).
Mentions: We checked for differences in miRNA distribution between whole milk, lipids and skim milk. In Fig 3, we represented in a graph the mean Cq with SEM of the six measured miRNAs in whole milk or its fractions. MiR-21, let-7d and let-7a Cq values were significantly lower in lipids than in skim milk (respectively p<0.001, p<0.001 and p<0.01) and than in full milk (respectively p<0.001, p<0.001 and p<0.01), whilst no differences were found between skim milk and whole milk. On the other hand, miR-16, let-7g and miR-146b Cq values were significantly higher in skim milk fractions than in lipids (respectively p<0.001, p<0.01 and p<0.01) and whole milk (respectively p<0.001, p<0.05 and p<0.001).

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