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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

Analysis of daily miRNA fluctuations in whole milk.Endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) have been analyzed in milk samples collected from four mothers (A to D) and expressed at different times throughout a day. A) Box-and-whisker plots (1–99 percentile) showing raw Cq data of all measured miRNAs. B) RefFinder analysis sustains let-7g/d as the best combination for normalization followed by let-7g/d/miR-146b. C) MiR-16, using both normalization factors (let-7g/d/miR-146b and let-7g/d), exhibits daily fluctuations in the milk from four mothers (p = 0.04). Data are obtained from milk collected from four healthy donors during one day (*p<0.05). D) MiR-21 and let-7a appear relatively stable over 24 hours.
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pone.0140488.g004: Analysis of daily miRNA fluctuations in whole milk.Endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) have been analyzed in milk samples collected from four mothers (A to D) and expressed at different times throughout a day. A) Box-and-whisker plots (1–99 percentile) showing raw Cq data of all measured miRNAs. B) RefFinder analysis sustains let-7g/d as the best combination for normalization followed by let-7g/d/miR-146b. C) MiR-16, using both normalization factors (let-7g/d/miR-146b and let-7g/d), exhibits daily fluctuations in the milk from four mothers (p = 0.04). Data are obtained from milk collected from four healthy donors during one day (*p<0.05). D) MiR-21 and let-7a appear relatively stable over 24 hours.

Mentions: As shown Fig 4, box plots (1–99 percentiles) have been again used to visualize raw Cq variations of the six measured miRNAs. MiR-146b, let-7d and let-7g appeared relatively stable (Cq SDs respectively were 0.9, 1.5 and 1.6), while miR-16, miR-21 and let-7a were more variable (Cq SD ≥ 2; Fig 4A). Because choosing the most stably expressed reference gene for normalization is not always recommended [34], we further evaluated the candidate references by RefFinder. RefFinder analyses established that let-7d, let-7g and miR-146b were the most stable references, and that the geometric means of let-7d and let-7g (let-7d/let-7g) gave the best stability value, followed by the combination of the three controls let-7d/let-7g/miR-146b (Fig 4B). We have tested the two suggested combinations, let-7g/d and let-7g/d/miR-146b, in normalizing miR-21, miR-16 and let-7a levels. Both allowed identification of miR-16 fluctuations throughout the 24H (p = 0.04). Once again, the use of three references is preferred to the use of two or only one reference27. Using the normalization factor let-7g/d/miR-146b, the variance was reduced and the statistical efficiency improved compared to the use of let-7g/d. We discovered higher miR-16 levels in evening milk, collected from 18:00 to 20:00, than in morning milk, expressed from 7:00 to 9:00 (p<0.05) (Fig 4C). MiR-21 and let-7a levels, in both normalization strategies used, appeared relatively stable over the 24H (Fig 4D). As we could not possibly obtain a similar baseline time for all the donors, the four mothers differ in miR-16 fluctuations in their milk (S5 Fig); moreover, an intra-individual variability was found (S6 Fig).


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)

Analysis of daily miRNA fluctuations in whole milk.Endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) have been analyzed in milk samples collected from four mothers (A to D) and expressed at different times throughout a day. A) Box-and-whisker plots (1–99 percentile) showing raw Cq data of all measured miRNAs. B) RefFinder analysis sustains let-7g/d as the best combination for normalization followed by let-7g/d/miR-146b. C) MiR-16, using both normalization factors (let-7g/d/miR-146b and let-7g/d), exhibits daily fluctuations in the milk from four mothers (p = 0.04). Data are obtained from milk collected from four healthy donors during one day (*p<0.05). D) MiR-21 and let-7a appear relatively stable over 24 hours.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608744&req=5

pone.0140488.g004: Analysis of daily miRNA fluctuations in whole milk.Endogenous references (let-7a, let-7g, let-7d, miR-16, miR-146b and miR-21) have been analyzed in milk samples collected from four mothers (A to D) and expressed at different times throughout a day. A) Box-and-whisker plots (1–99 percentile) showing raw Cq data of all measured miRNAs. B) RefFinder analysis sustains let-7g/d as the best combination for normalization followed by let-7g/d/miR-146b. C) MiR-16, using both normalization factors (let-7g/d/miR-146b and let-7g/d), exhibits daily fluctuations in the milk from four mothers (p = 0.04). Data are obtained from milk collected from four healthy donors during one day (*p<0.05). D) MiR-21 and let-7a appear relatively stable over 24 hours.
Mentions: As shown Fig 4, box plots (1–99 percentiles) have been again used to visualize raw Cq variations of the six measured miRNAs. MiR-146b, let-7d and let-7g appeared relatively stable (Cq SDs respectively were 0.9, 1.5 and 1.6), while miR-16, miR-21 and let-7a were more variable (Cq SD ≥ 2; Fig 4A). Because choosing the most stably expressed reference gene for normalization is not always recommended [34], we further evaluated the candidate references by RefFinder. RefFinder analyses established that let-7d, let-7g and miR-146b were the most stable references, and that the geometric means of let-7d and let-7g (let-7d/let-7g) gave the best stability value, followed by the combination of the three controls let-7d/let-7g/miR-146b (Fig 4B). We have tested the two suggested combinations, let-7g/d and let-7g/d/miR-146b, in normalizing miR-21, miR-16 and let-7a levels. Both allowed identification of miR-16 fluctuations throughout the 24H (p = 0.04). Once again, the use of three references is preferred to the use of two or only one reference27. Using the normalization factor let-7g/d/miR-146b, the variance was reduced and the statistical efficiency improved compared to the use of let-7g/d. We discovered higher miR-16 levels in evening milk, collected from 18:00 to 20:00, than in morning milk, expressed from 7:00 to 9:00 (p<0.05) (Fig 4C). MiR-21 and let-7a levels, in both normalization strategies used, appeared relatively stable over the 24H (Fig 4D). As we could not possibly obtain a similar baseline time for all the donors, the four mothers differ in miR-16 fluctuations in their milk (S5 Fig); moreover, an intra-individual variability was found (S6 Fig).

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