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Stability of circulating microRNAs in serum.

Author(s): Kupec T, Bleilevens A, Iborra S, Najjari L, Wittenborn J, Maurer J, Stickeler E

Publication: PLoS One, 2022, Vol. 17, Page e0268958

PubMed ID: 36044434 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the levels of sixteen miRNA in serum collected after healthy volunteers fasted overnight and then again at noon after they had eaten, and between miRNA extracted immediately after collection and after serum was stored for 14 days at -80°C before RNA isolation. Blood was collected from eight healthy volunteers first thing in the morning after an overnight fast and at noon after eating. Specimens were allowed to clot before separation of serum by centrifugation at 2500 g for 10 min. miRNA was isolated from serum aliquots on the day of collection and after 14 days at -80°C using the miRNeasy Mini Kit. Isolated miRNAs were stored at -80°C. miRNAs were reverse transcribed with the TaqMan Advanced cDNA Synthesis Kit with the ath-miR-159a exogenous spike-in control added during the poly-A-tailing step.  Levels of miR-361-5p, miR-28-5p, miR-194-5p, miR-125b-5p, miR-192-5p, miR-27b-3p, miR-151a-5p, miR-1260a, miR-100-5p, miR-151a-3p, miR-323b-5p, miR-99a-5p, miR-28-3p, miR-22-3p, miR-193a-5p, miR-193b-3p and the spike-in control were quantified using TaqMan real-time PCR assays.  Real-time PCR quantification cycles values were normalized to ath-miR-159a.  Significance was set to P<0.01.

   Summary of Findings:

   Levels of the sixteen analyzed miRNA were comparable in morning fasting specimens and those obtained at noon after eating. When serum was stored for 14 days at -80°C, all sixteen miRNAs were detected at lower levels than when processed immediately.  However, differences in miRNA levels between aliquots processed immediately and those stored for 14 days at -80°C were not significant for specimens collected first thing in the morning and significance was limited to miR-22-3p (P=0.0069) in specimens collected at noon. Interestingly, a more than one cycle difference in miR-193b-3p was found between aliquots processed immediately and those stored for 14 days at -80°C; serum levels of miR-193b-3p were higher in fresh specimens when collected in the morning, but were higher in frozen aliquots when collected at noon.

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Time of biospecimen collection	Fasting in morning Noon
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Storage	Storage duration	<1 day 14 days
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-361-5p miR-28-5p miR-194-5p miR-125b-5p miR-192-5p miR-1260a miR-100-5p miR-323b-5p miR-99a-5p miR-28-3p miR-22-3p miR-193a-5p miR-193b-3p ath-miR-159a miR-27b-3p miR-151a-3p

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