Stability of microRNAs in serum and plasma reveal promise as a circulating biomarker.
Author(s): Katayama ES, Hue JJ, Loftus AW, Ali SA, Graor HJ, Rothermel LD, Londin E, Zarei M, Winter JM
Publication: Noncoding RNA Res, 2025, Vol. 15, Page 132-141
PubMed ID: 40926836 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of microRNAs (miRNAs) in (i) plasma and serum that were stored post-separation at room temperature or on wet ice for up to 24 h and in (ii) plasma from blood that was stored at room temperature up to 6 h prior to plasma separation. Effects of plasma and serum storage were investigated by real-time PCR analysis of 5 miRNAs and two housekeeping RNAs in specimens from five volunteers and by next-generation sequencing (NGS) using the plasma of two volunteers. Effects of storing blood for up to 6 h prior to plasma separation on miRNA levels were investigated by NGS of plasma from a single volunteer.
Conclusion of Paper
Levels of 18S rRNA and β-actin declined progressively during plasma storage at room temperature, with significantly lower levels observed after ≥2 h of plasma storage (P<0.01, all); however, all five miRNA targets were unaffected by storage of plasma or serum on ice or at room temperature for up to 24 h before extraction. NGS revealed changes in the levels of a small number of miRNAs (<1%) over the course of plasma storage at room temperature. However, affected miRNAs did not xxhibit a common pattern of change in response to storage, as individual miRNAs differed between volunteers; further, the low expression levels suggest that storage-associated differences in miRNA may in fact be due to background noise.
When blood from a single volunteer was stored at room temperature prior to plasma separation, one miRNA decreased and one increased over the course of 2 h, and six decreased and one increased when blood was stored for 6 h.
Studies
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Study Purpose
This study compared levels of microRNAs (miRNAs) in plasma that was stored at room temperature or on wet ice for up to 24 h. Blood was collected from five healthy volunteers (2 women and 3 men) into K2EDTA tubes and serum clotting tubes. Plasma/serum were separated by centrifugation at 1200 g for 10 min at room temperature, followed by 1500 g for an additional 5 min. Plasma and serum aliquots were stored at room temperature and on ice for 0, 2, 6, and 24 h before miRNA isolation with the Qiagen miRNeasy Serum/Plasma Kit. miR-15b, miR-16, miR-21, miR-24, and miR-223 as well as 18S rRNA and β-actin were quantified by real-time PCR.
Summary of Findings:
Transcript levels of 18S rRNA and β-actin declined progressively when plasma was stored at room temperature; significantly lower levels of 18S rRNA (20%) and β-actin (30%) were observed after 2 h of plasma storage at room temperature (P<0.01, both), decreasing by 77% and 57% lower, respectively, after 24 h of storage (P<0.01, both). In contrast, all five miRNAs were unaffected by storage of plasma or serum on ice or at room temperature for up to 24 h before extraction.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Storage Storage duration 0 h
2 h
6 h
24 h
Storage Storage temperature Room temperature
On ice
Real-time qRT-PCR Specific Targeted nucleic acid miR-15b
miR-16
miR-21
miR-24
miR-223
18S rRNA
β-actin
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Study Purpose
This study compared levels of microRNAs (miRNAs) in plasma that was stored at room temperature for up to 24 h and in plasma isolated from blood that was stored at room temperature for up to 6 h prior to plasma separation. Blood was collected from three healthy volunteers into K2EDTA tubes. Blood from one volunteer was stored at room temperature for 0, 2 and 6 h before plasma separation, while plasma was obtained from the blood of the other two volunteers within 30 min. Plasma was separated by centrifugation at 1200 g for 10 min followed by 1500 g for 5 min. Plasma aliquots from the promptly processed blood were stored at room temperature for 0, 6, and 24 h before miRNA isolation. RNA was isolated with the Qiagen miRNeasy Serum/Plasma Kit and evaluated with a fluorometer and a TapeStation. Sequencing libraries were constructed using the Illumina TruSeq Small RNA library Prep Kit and pair-end sequenced on an Illumina HiSeq instrument.
Summary of Findings:
Approximately 650 miRNAs were detected via sequencing, with an extremely small number of miRNAs affected by plasma storage at room temperature: 6 h of storage, 2 or 3 miRNAs decreased and 0 or 4 increased (2 donor); 24 h of storage, 0 or 5 decreased and 5 or 8 increased when stored for 24 h (2 donors). Overall, 99.2% and 99% of the detected miRNAs were stable when plasma was stored post separation for 6 h and 24 h, respectively, at room temperature. Importantly, the miRNAs that were affected did not show a distinct pattern of change among timepoints, differing between the two volunteers; notably, all affected miRNAs had low counts per million. The authors state this indicates that the differences were due to background noise rather than true changes.
When blood from a single volunteer was stored at room temperature prior to plasma separation, one miRNA decreased and one increased when stored for 2 h, and six decreased and one increased when stored for 6 h.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage conditions Blood
Plasma
Storage Storage duration 0 h
2 h
6 h
24 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated