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Impact of long-term storage and freeze-thawing on eight circulating microRNAs in plasma samples.

Author(s): Matias-Garcia PR, Wilson R, Mussack V, Reischl E, Waldenberger M, Gieger C, Anton G, Peters A, Kuehn-Steven A

Publication: PLoS One, 2020, Vol. 15, Page e0227648

PubMed ID: 31935258 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of long-term storage of EDTA plasma on levels of eight miRNAs. Differences due to patient BMI, age, and sex on the same eight miRNAs were also investigated. EDTA plasma was collected from 10 non-smoking, non-diabetic women (25-30 years) in the Cooperative Health Research in the Region of Augsburg (KORA) study and stored 2-17 years in vapor-phase liquid nitrogen (-180°C). Importantly, specimens were obtained from the same 10 women at each of the three different time-points (1999-2001, 2006-2008, and 2013-2014) and each specimen was analyzed once rather than sampled once and analyzed at three time-points. Thus, all effects could be attributable to patient aging, changes in methodology, or other changes rather than storage. To investigate the effects of patient BMI, age. and sex on levels of miRNA, EDTA plasma from all 300 patients in the KORA study (male and females) were investigated. miRNA was extracted from plasma using the miRCURY RNA Isolation Kit–Biofluids with MS2 carrier RNA and reverse-transcribed using the Exiqon A/S Universal cDNA Synthesis Kit II. miRNAs were quantified using two miRCURY LNA qPCR panels. Hemolysis was assessed by the ratio of miR-451a and miR-23a-3p.

   Summary of Findings:

   Power analysis showed the minimal detectable significant effect (P<0.05) would be a difference of means of 0.58, which the authors state is a large difference. Principal cluster analysis (PCA) showed some clustering in component 2 based on storage duration but did not cluster by patient. Only UniSp6 normalized miR-451a levels were significantly affected by storage duration or patient aging (ANOVA P-value=0.0062).  UniSp6 normalized miR-451a levels were significantly lower in specimens stored for 17 years than those from the same patients collected 15 years later and stored for 2 years (P<0.001), but differences in levels between specimens stored for 9 years versus those collected from the same patients 7 years later and stored for 2 years were non-significant (P=0.47). Importantly, there was no association of any of the miRNAs (including miR-451a) with patient BMI, age, or sex in the cohort of 300 specimens. As miR-451a is a marker of red blood cell contamination, the authors state the difference in levels could also reflect differences in sampling (such as cannulas used or changes in freezing procedure) over the study period rather than effects of frozen storage.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Time of biospecimen collection	1999-2001 2006-2008 2013-2014
   Preaquisition	Patient gender	Male Female
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-103a-3p miR-191-5p miR-451a miR-30c-5p miR-93-5p miR-23a-3p miR-24-3p miR-33b-5p
   Preaquisition	Patient body mass index	Unspecified range
   Storage	Storage duration	2 years 9 years 17 years
   Preaquisition	Patient age	Unspecified range

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2. Study Purpose

   The purpose of this study was to investigate the effects of freeze/thaw cycling of EDTA plasma on levels of eight miRNAs. EDTA blood was collected from 6 non-smoking, non-diabetic women (25-30 years) and processed to plasma according to the KORA SOPs. Plasma was aliquoted and stored at -80°C for 4 weeks before analysis. Specimens were then thawed on ice, miRNA was extracted and analyzed, and remaining plasma was snap-frozen in liquid nitrogen and subjected to up to four freeze-thaw cycles. miRNA was extracted from plasma using the miRCURY RNA Isolation Kit–Biofluids with MS2 carrier RNA and reverse-transcribed using the Exiqon A/S Universal cDNA Synthesis Kit II. miRNAs were quantified using two miRCURY LNA qPCR panels. Hemolysis was assessed by the ratio of miR-451a and miR-23a-3p.

   Summary of Findings:

   PCA revealed some clustering based on the number of freeze/thaw cycles and ANOVA analysis revealed a significant effect of freeze/thaw cycling on UniSp6-normalized levels of miR-30c-5p (P=0.00055), but the authors state the study was powered sufficiently only to detect large effects (difference of means of >0.76). Significant differences in miR-30c-5p levels were revealed between specimens freeze/thaw cycled three or four times versus once (P<0.001, both) or twice (P=0.0019 and 0.00154, respectively).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-103a-3p miR-191-5p miR-451a miR-30c-5p miR-93-5p miR-23a-3p miR-24-3p miR-33b-5p
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles 4 cycles

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