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Whole Blood Holding Time Prior to Plasma Processing Alters microRNA Expression Profile.

Author(s): Kim SH, MacIntyre DA, Sykes L, Arianoglou M, Bennett PR, Terzidou V

Publication: Front Genet, 2021, Vol. 12, Page 818334

PubMed ID: 35096023 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the potential impact of delays to centrifugation on the degree of hemolysis and microRNA (miR, miRNA) expression levels in matched plasma from pregnant women. EDTA whole blood was collected from five pregnant women and stored at 4°C for 30 min, 2 h, 6 h or 24 h before centrifugation. Plasma was separated by centrifugation at 3000 g for 10 min at 4°C and stored frozen at -80°C until extraction. RNA was extracted using the Norgen Plasma/Serum Circulating and Exosomal RNA Purification Kit, with cel-254 added during the lysis step. RNA was further purified using Ultra YM-3 columns before reverse transcription with the miRCURY LNA Universal RT miRNA cDNA synthesis kit II including the UniSp6 spike-in control. Levels of 179 miRNAs were quantified by real-time PCR using the Serum/Plasma Focus miRNA PCR panel. Hemolysis was assessed by spectrophotometer with a value >0.2 considered hemolyzed. Hemolysis was also assessed by real-time PCR amplification of miR-451a and hsa-miR-23a-3p, with moderate and high hemolysis defined at >5 and >7 cycle smaller CT values for miR-451a than hsa-miR-23a-3p, respectively.

   Summary of Findings:

   As expected, hemolysis increased with longer delays to plasma isolation. By both methods some specimens were considered hemolyzed after only a 2 h delay. While the average miR ratio was significantly higher starting after only 2 h (P<0.01), the absorbance at 414 nm was only significantly higher after ≥6 h (P<0.05).  53 of 179 miRNAs were differentially affected by processing delays in ANOVA analysis and these included miR-16-5p, miR-25-3p and miR-223-3p which are often used as controls and miR-150-5p, miR-191-5p and miR-29a-3p which were previously found to be associated with preterm birth, small for gestational age births, and preeclampsia. The five miRNAs with the largest changes after processing delays (miR-342-3p, miR-150-5p, let-7a-5p, miR-486-5p, miR-451a) all showed decreased Cq (increased levels) with longer times. The change in miR-451a with longer centrifugation delays was remarkably consistent among patients. However, PCA clustering based on miRNA profiles showed clear differences based on delayed blood processing with differences observed after only 2 h, but the differences differed among the specimens analyzed.  Six miRNAs were found to be significantly associated with hemolysis, four of them negatively (miR-874-3p, miR-335-3p, miR-425-5p, and miR-199a-5p) and two positively (miR-16-5p and miR-451a). Of the miRNA associated with hemolysis, only miR-451a was also affected by holding time.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   Protein	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Storage duration	30 min 2 h 6 h 24 h
   Spectrophotometry Specific	Technology platform	Hemolysis measured by spectrophotometer Hemolysis measured by real-time PCR
   Biospecimen Aliquots and Components	Hemolysis	Absent Present Quantitative measure

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