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MiRNA profiles in blood plasma from mother-child duos in human biobanks and the implication of sample quality: Circulating miRNAs as potential early markers of child health.

Author(s): Dypås LB, Gützkow KB, Olsen AK, Duale N

Publication: PLoS One, 2020, Vol. 15, Page e0231040

PubMed ID: 32240265 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared markers of plasma quality and miRNA profiles in maternal and cord blood plasma. Paired maternal and cord blood were collected into CPT tubes from 38 women giving birth at the Oslo University Hospital. Plasma was obtained by centrifugation at 1700 x g for 30 min, aliquoted, and stored frozen at -20°C. Thawed plasma (details of thawing not provided) were centrifuged for 5 min at 16000 x g at 4°C. Subsequently, plasma quality was evaluated and RNA was extracted from the supernatant using the miRNeasy Serum/Plasma Kit with the addition of MS2 carrier to the lysis and three spike-in C. elegans miRNAs (cel-miR-39, cel miR-45, and cel-miR-238). Extracted RNA was stored at -80°C. RNA was reverse-transcribed using the miScript II RT Kit stored at -20°C until real-time PCR amplification of 204 miRNAs using miScript SYBR Green PCR Kit. Plasma quality was assessed using a CASY Cell Counter and Analyzer Model TT and by determination of hemolysis by spectrophotometry and using the ratio of miR-451 to miR-23a.

   Summary of Findings:

   Generally, plasma was shown to be of high quality with few particles >2 µm, indicating an absence of cells and platelets. Peaks at around 1 µm were observed in both specimen types with larger and more variable peaks in maternal plasma. The authors state these peaks correspond to the size of microparticles. Spectrophotometric analysis showed similar and low degree of hemolysis in both the cord blood plasma (mean 0.18 ±0.015, range: 0.05–0.44) and the maternal plasma (mean 0.25 ±0.020, range: 0.03–0.48). Similarly, hemolysis as assessed by real-time PCR ratios of miR-451 to miR-23a was less than the threshold of 5, indicating low levels of hemolysis in both specimen types (mean 2.78 ±0.17 in cord blood and 2.50 ±0.19 in maternal blood). Of the 201 miRNAs assessed, 61 passed quality assurance filtering and 37 of these were found to be significantly different in plasma than cord blood. After correction for multiple testing and eliminating those with fold-change < ±1.5, 19 miRNAs remained and all were higher in cord blood plasma than maternal plasma. Correlations in miRNA levels were weak between maternal and cord blood plasma when all specimens were considered (R²=0.2274, P<0.0001) and after removal of three specimens with unusually high expression (R²=0.2842, P<0.0001). Analysis of individual miRNAs revealed the strongest correlations were in miR-142-3p levels (R²=0.637, P<0.0001).

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Cell count/volume	Real-time qPCR
   Cell count/volume	Spectrophotometry
   Cell count/volume	Hematology/ auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Cord blood Venous blood
   Spectrophotometry Specific	Technology platform	Real-time PCR
   Biospecimen Aliquots and Components	Blood and blood products	Umbilical cord blood Plasma

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