Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Assay reproducibility in clinical studies of plasma miRNA.

Author(s): Rice J, Roberts H, Burton J, Pan J, States V, Rai SN, Galandiuk S

Publication: PLoS One, 2015, Vol. 10, Page e0121948

PubMed ID: 25853871 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 delayed isolation of plasma, time of blood collection, inter- and intra- operator variability, and extraction method used on microRNA levels in plasma. The authors also reviewed the literature published between July 1, 2013 and June 30, 2014 to identify variables in preanalytic methods used for the analysis of miRNA in plasma. EDTA blood was obtained via venipuncture from 16 patients with colorectal advanced adenomas and 16 age, gender, and race matched patients without colorectal neoplasia (control). Unless otherwise specified, blood was stored at 4°C for up to 24 h before plasma isolation by centrifugation at 600 relative centrifugal force for 15 minutes. Plasma was stored at -80°C until extraction of miRNA using a phenol/guanidine-based lysis with the miRNeasy Kit. RNA was quantified using a NanoDrop spectrophotometer. RNA was reverse-transcribed using TaqMan miRNA reverse transcription kit and amplified using the Step-One Plus RT-PCR System. The effects of delayed centrifugation were investigated by storing the blood from five control patients for 30 min, 12 h, 24 h, 48 h, or 72 h before plasma isolation. To investigate the effect of time of acquisition, blood was drawn from control patients at 6:30 AM and 6:30 PM on the same day and plasma was isolated within 30 min. The effects of RNA extraction method were investigated by extracting RNA from aliquots of the same plasma specimen using Trizol LS reagent protocol followed by an extended overnight drying period or purification using a modified miRNeasy protocol and preamplified using the Applied Biosystems protocol for preamplification for TaqMan miRNA assays.

   Summary of Findings:

   None of the miRNAs investigated were significantly altered by refrigerated storage of blood for 12 h rather than 30 min before plasma isolation. Compared to plasma isolated within 30 min of blood collection, plasma isolated after 24 h had 2.84-fold higher levels of miR-122 (P<0.001); plasma isolated after 48 h had 4.47-fold higher levels of miR-122 (P<0.001), 1.69-fold higher levels of miR-485-3p (P=0.02), and 1.48-fold higher levels of miR-21 ( P=0.03); and plasma isolated after 72 h had 5.94-fold higher levels of miR-122 (P<0.001), 2.22-fold higher levels of miR-485-3p (P<0.001), 1.26-fold higher levels of miR-21 ( P=0.05), 2.56-fold higher levels of miR-218 (P<0.001), and 2.29-fold higher levels of miR-523 (P=0.04). However, levels of miR-374, miR-142-3p, miR-374-5p, miR-376c, miR-27a, and miR-520d-5p were not significantly different in specimens refrigerated for up to 72 h before centrifugation than in immediately processed specimens. Highlighting the importance of these findings, the authors found during review of the literature published between July 1, 2013 and June 30, 2014 that 59.6% of the studies isolated plasma within 2 h of phlebotomy, 10.8% isolated plasma 2 or more hours after phlebotomy, and 29.7% did not indicate the duration of storage. None of the miRNAs were significantly different between specimens collected at 6:30 AM and those collected from the same individuals 12 h later. Further, there were no significant differences between duplicates. However, miR-21 levels in Trizol-extracted specimens and miR-485-3p and miR-21 in miRNeasy-extracted specimens differed significantly when reverse transcription and preamplification were performed separately by two different people, but the authors believe the differences were within the range of experimental error. Using ANOVA, the authors found that the inter-operator variability did not affect the ability to find differences between groups. The yield of RNA was higher using the miRNeasy than Trizol method and, despite inclusion of a preamplification step with the Trizol method, 15% (109 of 704) of miRNAs were not detected while only 1 of 704 were not detected when extraction was with miRNeasy. Importantly, the authors literature review found Trizol was used for extraction in 17.6% of studies, miRNeasy in 33.8% of studies, and mirVana in 40.5% of studies.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified
   • Neoplastic - Carcinoma

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Time of biospecimen collection	6:30 AM 6:30 PM
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Storage duration	30 min 12 h 24 h 48 h 72 h
   Analyte Extraction and Purification	Analyte purification	miRNeasy Trizol

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