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Impact of ambient temperature exposure on miRNA stability in human plasma.

Author(s): Desgagné V, Lavoie F, Soukar I, Hivert MF, Bouchard L, O'Tierney-Ginn PF

Publication: Biotechniques, 2025, Vol. , Page 1-12

PubMed ID: 40916661 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared levels of six miRNAs by digital PCR in archival plasma that had not been thawed with plasma samples that were thawed and stored at room temperature for 24, 48 or 72 h before refreezing. Effects of elution order, extraction batch and dPCR plate on levels of the spike-in extraction control were also investigated. EDTA blood was collected from nine pregnant women in the first trimester of pregnancy after a 1 hour glucose challenge. Plasma was separated within 30 min by centrifugation at 2500 x g for 10 min at 4°C. Plasma aliquots were stored at -80°C for 12 years. A single aliquot from each participant was continuously stored at -80°C until RNA extraction, and three aliquots were thawed in a room temperature cooling microtube holder and pooled (plasma from 3 women per pool). Thawed, pooled plasma was realiquoted, and matched aliquots were stored at room temperature (20°C) for 24, 48 and 72 h before refreezing. Plasma was spiked with cel-miR-39-3p, and RNA was extracted using the miRVana PARIS Kit. Isolated RNA was stored at -80°C. Levels of cel-miR-39-3p, let-7b-3p, miR144-5p, miR-150-5p, miR-517a-3p, miR-524-5p, and miR-1283 were quantified by miRCURY LNA miRNA PCR Assays on a QIAcuity ONE5 Plex Digital PCR System with UniSp6 used as a reverse transcription spike-in control. miRNA levels were normalized to cel-miR-39-3p.

   Summary of Findings:

   Levels of the extraction spike-in control (cel-miR-39-3p) were highly variable (39%), although the reverse transcription control (UniSp6) had much less variability (5.2%). Levels of cel-miR-39-3p were negatively correlated with the order of elution (r_s = −0.522, P=0.001) and differed between the two extraction batches (P=0.011) but not dPCR plate. The miRNAs of interest were expressed at very different levels in plasma, with approximately 150-fold and 100-fold higher expression of miR-150-5p than miR-517a-3p and miR-1283, respectively.  The miRNAs with the lowest expression (miR-1283 and miR-517a-3p) also had very high Poisson confidence intervals at 95% (86.3 ± 36.5% and 82.9 ± 57.3%, respectively), while the Poisson confidence intervals at 95% were low for the highly expressed miR-150-5p (9.3 ± 1.2%). The average absolute concentration of each of the miRNAs was lower in specimens that were thawed for 72 h at room temperature compared to those stored continuously at -80°C; and levels of five miRNAs (not miR-1283) were lower in specimens that were thawed for 48 h than those that remained frozen (not thawed). When normalized to cel-miR-39-3p, no significant effect of thawing/room temperature storage on miRNA levels was observed, but a clear trend toward lower levels in specimens subjected to thawing was observed. This trend toward lower miRNA levels with thawing and room temperature storage was dependent on miRNA species, with only an 18% reduction observed in let-7b-3p levels after a 72 h thaw, but 52%, 86% and 77% reductions in miR-517a-3p observed after 24, 48 and 72 h thaw durations, respectively. No correlation was found between the GC content or abundance of the miRNA and its stability.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   RNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Post-thaw duration	0 h 24 h 48 h 72 h
   Digital PCR Specific	Targeted nucleic acid	let-7b-3p miR-150-5p miR-517a-3p miR-524-5p miR-1283 miR-144-5p cel-miR-39-3p (extraction spike-in control) UniSp6 (reverse transcription spike-in control)
   Analyte Extraction and Purification	Analyte isolation method	Effect of extraction batch and elution order on spike-in control investigated
   Storage	Freeze/thaw cycling	0 cycles 1 cycle

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