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Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance.

Author(s): Leidinger P, Backes C, Rheinheimer S, Keller A, Meese E

Publication: PLoS One, 2015, Vol. 10, Page e0143321

PubMed ID: 26599228 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared miRNA expression in blood collected and stored in PAXgene tubes with blood collected in K₂EDTA tubes and stored for 0 min, 10 min, or 2 h before transfer to PAXgene tubes. Blood was collected from six healthy volunteers into K₂EDTA and PAXgene Blood RNA tubes. Blood from the K₂EDTA tubes was transferred into the PAXgene tubes after 0 min, 10 min, and 2 h and stored at room temperature. Two hours after the last transfer, all tubes were placed in storage at -20°C until RNA isolation using the PAXgene miRNA Blood Kit and a QIAcube. RNA concentration was measured using a NanoDrop-2000 and quality/integrity assessed using a Bioanalyzer with Agilent 6000 RNA Pico Chips. miRNA levels were quantified using SurePrint G3 8x60K miRNA microarrays. Identified tube-type specific changes in miRNA levels were confirmed using Real-Time PCR and the miScript PCR System.

   Summary of Findings:

   RNA yields and integrity were comparable among the specimens, regardless of duration of storage in K₂EDTA tubes. A total of 201 miRNAs were detected in all specimens, 806 were not detected in any of the specimens, and 399 were detected in one or more specimen. Importantly, there were no significant differences in the mean number of miRNAs detected between the groups (288 miRNAs in the PAXgene specimen and 288, 278, and 279 miRNAs in the specimens stored in K₂EDTA tube for 0 min, 10 min, and 2 h; respectively, before transfer to PAXgene). An investigation of differences in miRNA presence between conditions identified 12 miRNAs; including miRNA-769-3p, let-7e, miR-23-c, miR-602, miR-30c-1, miR-337-3p, miR-129, miR-3646, miR-1288, and miR-3907. The largest difference in abundance was found for miR-769-3p which was found in all specimens stored in K₂EDTA tubes for 2 h before transfer but in none of the specimens collected in PAXgene tubes. Heatmaps of the 321 miRNAs detected in at least 25% of specimens mostly clustered specimens based on patient and no clustering based on condition was observed. ANOVA analysis identified differences in abundance in miR-769-3p among the conditions (adjusted P=0.003) with highest levels observed in specimens stored for 2 h in K₂EDTA tubes. The average coefficient of variance (CV) in miRNA levels was lowest for specimens collected in K₂EDTA tubes and immediately transferred and highest in specimens collected in PAXgene tubes (9.4% versus 10.7%, P=0.0045). A higher average CV was observed for miR-769-3p and miR-135a than CV within condition; indicating an effect of processing on their expression. Pairwise comparisons revealed 33, 22, and 41 miRNAs were significantly different in PAXgene specimens from those collected and stored in K₂EDTA for 0 min, 10 min, and 2h, respectively; 14 and 15 miRNAs in specimens stored in K₂EDTA tubes for 0 min versus 10 min and 2 h, respectively; and 44 miRNAs in specimens stored in K₂EDTA tubes for 10 min versus 2 h. However, the only differences that remained significant between the specimens stored for 2 h in K₂EDTA tubes and those collected in PAXgene tubes after correction for multiple testing were for miR-769-3p, miR-4257, miR-4270, and miR-1915. Real-time PCR confirmed significantly higher miR-769-3p in specimens stored in K₂EDTA tubes for 2 h than in specimens collected in PAXgene tubes (P=0.004), but also significantly higher levels in blood stored in K₂EDTA tubes for 10 min than in specimens collected in PAXgene tubes (P=0.003). A trend toward higher miR-1915 and hsa-miR-4257 levels in specimens stored in K₂EDTA tubes for 2 h than in specimens collected in PAXgene tubes was observed by real-time PCR but was not significant. In contrast, real-time PCR did not confirm any differences in miR-4270 levels between groups.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   RNA	DNA microarray
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 min 10 min 2 h
   Biospecimen Acquisition	Type of collection container/solution	PAXgene tube K2EDTA tube

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