Impact of cellular miRNAs on circulating miRNA biomarker signatures.

Author(s): Duttagupta R, Jiang R, Gollub J, Getts RC, Jones KW

Publication: PLoS One, 2011, Vol. 6, Page e20769

PubMed ID: 21698099 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of patient gender, blood component, and centrifugation steps (one, two or three) on levels of cellular miRNAs and cell free CNAs. Blood was collected in sodium EDTA tubes, stored on ice, and processed within 4 hours of collection. In addition to the buffy coat, leukocytes, red blood cells, and plasma fractions, plasma was subjected to further centrifugation to obtain additional supernatant (S1 and S2) and pellet (P1 and P2) fractions. Total miRNA was extracted using Trizol and mirVANA filters. Specimens from 17 males were used for the blood component analysis and specimens from 10 females and 8 males were used for the patient gender analysis.

   Summary of Findings:

   Gel electrophoresis showed good quality miRNA extraction from each of the 4 blood components: (plasma, buffy coat, red blood cells, leukocytes), and the 4 plasma fractions (S1, P1, S2, and P2). There were higher Affymetrix miRNA array signal intensities among the cellular fractions than any of the plasma, supernatant, or pellet fractions. The plasma fraction (centrifuged once) and both plasma pellet fractions (P1 and P2) had higher array signal intensities than either of the plasma supernatant fractions (S1 or S2). It was found that circulating miRNAs were enriched in S1 compared to the initial plasma fraction, but that a third centrifugations step to produce S2 did not further enrich circulating miRNAs due to the strong agreement in microarray data between S1 and S2 fractions. Importantly, there were no significant differences in the expression levels of circulating miRNAs between the initial plasma and S1 fractions. Less variability and more homogenous expression was shown across replicates for the S1 fractions compared to the initial plasma fraction. Analysis of 534 miRNAs revealed gender-specific expression differences in 4 (hsa-miR-548a-3p, hsa-miR-1323, hsa-miR-940, and hsa-miR-1292). In all 4 cases, expression was significantly higher (1.63 to 1.94-fold) in specimens from females. The increased expression of hsa-miR-1323 and hsa-miR-1292 in plasma from females was confirmed by real time qRT-PCR.

   Biospecimens

   • Bodily Fluid - Buffy Coat
   • Cell - Red Blood Cells
   • Cell - White Cells
   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Biospecimen Aliquots and Components	Blood and blood products	Buffy coat Leukocyte Plasma Red blood cells
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared

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