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miRNAs in platelet-poor blood plasma and purified RNA are highly stable: a confirmatory study.

Author(s): Muth DC, Powell BH, Zhao Z, Witwer KW

Publication: BMC Res Notes, 2018, Vol. 11, Page 273

PubMed ID: 29728133 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The study investigated the effects of analyzing PRP versus PPP, freeze-thaw cycling, storage of PPP and PRP, and storage of extracted RNA on the stability of miRNA. Blood was collected from two (freeze-thaw and plasma storage) or three (Extracted RNA storage) patients into syringes containing acid citrate dextrose. Blood was centrifuged at 1300 x g for 15 min within 15 min of collection. The supernatant (platelet-rich plasma, PRP) was aliquoted and some aliquots were centrifuged twice at 2500 x g for 15 min to produce platelet-poor plasma (PPP). RNA was extracted from two specimens of matched fresh PRP and PPP as well as from PRP and PPP that were freeze-thaw cycled (-80˚C to 22˚C) one, two, three, four, five, or six times or stored at 22˚C for 24 h. RNA was extracted using an Exiqon Biofluids Kit with glycogen as a carrier and stored at -80˚C until use. Extracted RNA was thawed and stored at room temperature for 0, 1, 3, or 7 days before analysis by real-time PCR. RNA was stem-loop reverse-transcribed and spiked with cel-miR-39-3p. Levels of miR-16-5p, miR-21-5p, and cel-miR-39-3p were quantified using Applied Biosystems real-time PCR assays.

   Summary of Findings:

   As expected, PRP had much higher levels of miR-16-5p and miR-21-5p and these differences were attributed to the platelets. Frozen PRP had higher CT values for both miRNAs than fresh PRP, but there did not appear to be further increases with more cycles and there was no effect of freeze-thaw cycling on the miRNAs in PPP. Similarly, a slight increase in miRNA CT values was observed when the PRP was stored for 24 h at room temperature before RNA extraction, but there was no effect of storing PPP for 24 h before RNA extraction. Extracted miR-16-5p was stable for 3 days at room temperature, but there was a trend toward higher CT values when stored for 7 days prior to analysis, although the difference was not significant.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Not specified

   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	Platelet-poor plasma Platelet-rich plasma
   Storage	Time at room temperature	0 h 24 h 0 days 1 day 3 days 7 days
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles 6 cycles
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)

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