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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

MiRNAs measured at different lactation periods in whole human preterm milk.Candidate endogenous references (let-7a, let-7g, let-7d, miR-16, miR-21 and miR-146b) were measured in mature human milk collected within month 2 of lactation, from 28 to 65 days postpartum. The Cq values of each miRNA are shown by box-and-whisker plots (1–99 percentile).
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pone.0140488.g001: MiRNAs measured at different lactation periods in whole human preterm milk.Candidate endogenous references (let-7a, let-7g, let-7d, miR-16, miR-21 and miR-146b) were measured in mature human milk collected within month 2 of lactation, from 28 to 65 days postpartum. The Cq values of each miRNA are shown by box-and-whisker plots (1–99 percentile).

Mentions: A simple and intuitive way to evaluate reference gene expression stability is the analysis of the variation; a higher variance is associated with less stability and poor normalization [32]. To determine the stability of six literature-based candidate miRNAs (miR-21, miR-16, let-7a, let-7g, let-7d) and miR-146b, we analyzed human breast milk (n = 15) collected within month 2 of lactation from four healthy mother donors (Table 1). Each sample represents a pool of milk collected during a single day of lactation from a single mother donor made by mixing equal quantities of breast milk collected from 2 to 5 time points during 24 hours. In that way, the clock time variations were masked in order to assess miRNA variations within month 2 of lactation. Raw Cq data of each candidate EC were grouped together and represented in box-and-whisker plots (1–99 percentile; Fig 1). These data revealed that miR-21 (mean Cq = 28.24, SD = 0.76) and miR-16 (mean Cq = 28.47, SD = 0.99) were highly stable during lactation in mature milk. MiR-146b was confirmed abundant and it also appeared relatively stable (mean Cq = 26.72, SD = 1.37).


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)

MiRNAs measured at different lactation periods in whole human preterm milk.Candidate endogenous references (let-7a, let-7g, let-7d, miR-16, miR-21 and miR-146b) were measured in mature human milk collected within month 2 of lactation, from 28 to 65 days postpartum. The Cq values of each miRNA are shown by box-and-whisker plots (1–99 percentile).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140488.g001: MiRNAs measured at different lactation periods in whole human preterm milk.Candidate endogenous references (let-7a, let-7g, let-7d, miR-16, miR-21 and miR-146b) were measured in mature human milk collected within month 2 of lactation, from 28 to 65 days postpartum. The Cq values of each miRNA are shown by box-and-whisker plots (1–99 percentile).
Mentions: A simple and intuitive way to evaluate reference gene expression stability is the analysis of the variation; a higher variance is associated with less stability and poor normalization [32]. To determine the stability of six literature-based candidate miRNAs (miR-21, miR-16, let-7a, let-7g, let-7d) and miR-146b, we analyzed human breast milk (n = 15) collected within month 2 of lactation from four healthy mother donors (Table 1). Each sample represents a pool of milk collected during a single day of lactation from a single mother donor made by mixing equal quantities of breast milk collected from 2 to 5 time points during 24 hours. In that way, the clock time variations were masked in order to assess miRNA variations within month 2 of lactation. Raw Cq data of each candidate EC were grouped together and represented in box-and-whisker plots (1–99 percentile; Fig 1). These data revealed that miR-21 (mean Cq = 28.24, SD = 0.76) and miR-16 (mean Cq = 28.47, SD = 0.99) were highly stable during lactation in mature milk. MiR-146b was confirmed abundant and it also appeared relatively stable (mean Cq = 26.72, SD = 1.37).

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