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Changes in the microRNA expression profile during blood storage.

Author(s): Haberberger A, Kirchner B, Riedmaier I, Henschler R, Wichmann C, Buhmann R, Pfaffl MW

Publication: BMJ Open Sport Exerc Med, 2018, Vol. 4, Page e000354

PubMed ID: 30018790 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate intra-individual variability in miRNA expression in whole blood and compare miRNA levels in fresh and stored ECs with those in whole blood collected 1, 2, or 3 weeks prior. Blood was obtained from 12 healthy, recreationally active male patients using a 20-gauge butterfly needle into PAXgene Blood RNA Tubes weekly for 3 weeks. One week following the last collection, blood was collected via the Composelect T3984-23 System and erythrocyte concentrates were isolated using the Compomat G4. An additive solution of phosphate, adenine, glucose, guanine, saline, and mannitol was added to the ECs and they were stored in PAXgene Blood RNA Tubes at 4°C for 1, 2, 4, or 6 weeks before sampling. RNA was extracted using the PAXgene Blood miRNA Kit and quantified using the RNA HS Assay Kit on a Qubit 2.0 Fluorometer. RNA integrity was evaluated using an Agilent 2100 and the RNA 6000 Nano and Small RNA Kits. RNA was stored at -80°C until library preparation. NGS libraries were constructed using the NEBNext Multiplex Small RNA Library Prep Set, purified using the MinElute PCR Purification Kit, and quantified using a DNA 1000 Chip. To ensure only small RNAs were included, libraries were size-separated on agarose gels and the MinElute Gel Extraction Kit was used to purify cDNAs between 135-160 bp. Purified cDNA was quantified using the Qubit ds HS DNA Assay Kit and sequenced using the TrueSeq SR Cluster Kit v3-cBOT-HS on a HiSeq 2000.

   Summary of Findings:

   The authors report that total RNA yield differed significantly between fresh and stored blood (P<0.01) but did not specify the direction or magnitude of the effect. The authors report no effect of storage or sampling group on the percentage of miRNA. No significant differences were identified when miRNA expression was compared between consecutive timepoints, but six miRNAs were found to be lower in blood specimens collected 1 week before EC collection than in blood collected 3 weeks before EC collection (P<0.05) and, based on this intra-individual variability, were excluded from further analysis. Combining the lists of the 20 most differentially regulated miRNAs in ECs compared to those in blood collected by venipuncture 1, 2, or 3 weeks prior resulted in a list of 22 miRNAs which were upregulated in the ECs. Similarly, 21 miRNAs were found to be on the lists of the 20 most upregulated miRNAs in ECs stored for 6 weeks versus blood collected 1, 2, or 3 weeks prior to erythrocyte collection. Storage of ECs in PAXgene tubes resulted in significant increases in miR-16-2-3p, miR-1260a, miR-1260b, miR-4443, miR-4695–3p, and miR-5100. Importantly, PCA separated both fresh and stored (6 weeks) ECs from blood collected 1 week prior to collection for ECs. 

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Time of biospecimen collection	3 weeks prior to ECs 2 weeks prior to ECs 1 weeks prior to ECs Whole blood collection for ECs
   Storage	Storage duration	0 weeks 1 weeks 2 weeks 4 weeks 6 weeks
   Biospecimen Aliquots and Components	Blood and blood products	Erythrocyte Whole blood

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