Study of the preanalytical variables affecting the measurement of clinically relevant free-circulating microRNAs: focus on sample matrix, platelet depletion, and storage conditions.
Author(s): Faraldi M, Sansoni V, Perego S, Gomarasca M, Kortas J, Ziemann E, Banfi G, Lombardi G
Publication: Biochem Med (Zagreb), 2020, Vol. 30, Page 010703
PubMed ID: 31839723 PubMed Review Paper? No
Purpose of Paper
This study compared miRNA detection and miRNA levels in plasma from K2EDTA tubes and plasma preparation tubes (PPT) and in K2EDTA platelet-poor plasma (PPP). The effects of immediately freezing the plasma or storing for 24 h at room temperature or 4°C before freezing were also investigated.
Conclusion of Paper
The number of miRNAs detected (cycle threshold value <37) depended on the plasma type with more miRNAs detected in the plasma obtained from the PPT tube than the K2EDTA plasma or PPP but no difference between the K2EDTA plasma or PPP when plasma frozen immediately. The average number of miRNAs up- or down- regulated between K2EDTA plasma and PPP was 5% and 7%, respectively; but was only 2% and 1% in PPT versus PPP, respectively. Storage at either temperature resulted in the detection of significantly more miRNAs in K2EDTA plasma, fewer miRNAs in PPP, and a comparable number of miRNAs in PPT plasma. Individual miRNAs were found to be up- or down-regulated after storage with a larger percentage affected in EDTA plasma (1-5%) than PPP (0-2%) or PPT (0-1%) plasma. Importantly, the authors state clinically-relevant miRNA levels were significantly affected by the different plasma types and storage temperatures/durations but the effects were tube type specific. None of the samples were found to have hemolysis as determined by a CT difference of >7 between miR-23a and miR-451.
Studies
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Study Purpose
This study compared miRNA detection and miRNA levels in plasma from K2EDTA tubes and plasma preparation tubes (PPT) and in K2EDTA platelet-poor plasma (PPP). The effects of immediately freezing the plasma or storing for 24 h at room temperature or 4°C before freezing were investigated. Venous blood was obtained from 10 healthy male volunteers into K2EDTA and PPT Vacutainers. Plasma was obtained by centrifugation at 1300 x g for 10 min at room temperature and transferred to new tubes. An aliquot of the plasma from the EDTA tube was recentrifuged at 2500 x g for 15 min to obtain platelet-poor plasma. Aliquots of PPT plasma and EDTA plasma (PPP and standard) were stored for 0 and 24 h at 4°C or room temperature before being frozen at -80°C. miRNA was isolated using the miRCURY RNA Isolation Kit and RT-PCR was performed using the miRCURY LNA Universal RT miRNA PCR Kit and the Serum/Plasma miRCURY LNA miRNA Focus Panel. All CT values were normalized to the global mean CT.
Summary of Findings:
Significantly more miRNAs were detected (cycle threshold value <37) in the plasma obtained from the PPT tube than the K2EDTA plasma (161 versus 96 of 179, P<0.001) or PPP (161 versus 103, P<0.001) when plasma was frozen immediately. PPP had slightly more miRNAs detected than plasma from the same K2EDTA tube (103 versus 96) but the difference was non-significant (NS). PPP also had lower levels of the platelet miRNAs miR-126-3p (2.78-fold, NS), miR-223-3p (1.61-fold, NS), and miR-27b-3p (2.58-fold, P=0.034). The average number of miRNAs up- or down- regulated in K2EDTA plasma versus PPP was 5% and 7%, respectively; but was only 2% and 1% in PPT versus PPP, respectively. Storage of K2EDTA plasma at either temperature resulted in the detection of significantly more miRNAs (P<0.001, both) with a larger number detected when stored at 4°C than RT (P=0.002). Additionally, storage resulted in increases in 1% (4°C) and 5% (RT) and decreases in 1% (4°C) and 4% (RT) of individual miRNAs in K2EDTA plasma. Storage of PPP from the EDTA tubes resulted in significantly fewer miRNAs being detected after 24 h at room temperature (P=0.002) or 4°C (P=0.004) but no differences between the storage temperatures. Storage resulted in increases in 0% (4°C) and 2% (RT) and decreases in 1% (both temperature) of individual miRNAs in PPP. The number of miRNAs detected in PPT plasma was unaffected by storage at either temperature but storage did result in increases in 0% (4°C) and 1% (RT) and decreases in 1% (4°C) and 0% (RT) of individual miRNAs. When stored for 24 h before freezing, the difference in the number of miRNAs detected in standard K2EDTA plasma and PPT disappeared at either 4°C (159 versus 157, NS) or room temperature (155 versus 136, NS), but the number of miRNAs detected in PPP decreased such that it was significantly lower after 24 h at either temperature than in standard K2EDTA plasma or PPT plasma (P<0.001, all). Importantly, the authors state that clinically-relevant miRNA levels were significantly affected by the different storage temperatures/durations in K2EDTA plasma (miR-125b-5p, miR-29b-3p, and miR-376a-3p), PPP plasma (miR-125a-5p), and PPT plasma (miR-125b-5p) and there were differences in levels among the plasma types for clinically-relevant miRNAs (miR-1, miR-125b-5p, miR-152-3p, miR-195a-5p, miR-210-3p, miR-375, and miR-378a-3p). None of the samples were found to have hemolysis as determined by a CT difference of >7 between miR-23a and miR-451.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Real-time qPCR RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution K2EDTA Vacutainer
PPT Vacutainer
Biospecimen Aliquots and Components Blood and blood products Plasma
Platelet-poor plasma
Storage Storage temperature 4°C
Room temperature
Storage Storage duration 0 h
24 h
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared