Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples

PLoS One. 2014 Nov 4;9(11):e111644. doi: 10.1371/journal.pone.0111644. eCollection 2014.

Abstract

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Blood Specimen Collection
  • Gene Expression Profiling
  • Humans
  • Oligonucleotide Array Sequence Analysis
  • Polymerase Chain Reaction
  • RNA Stability
  • RNA, Messenger / blood*
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification

Substances

  • RNA, Messenger

Grant support

All authors were funded by the FP7 SPIDIA (Standardization and improvement of generic pre-analytical tools and procedures for in vitro diagnostic) supported by European Union (www.spidia.eu). VK, DS and M. Kubista were supported by BIOCEV CZ.1.05/1.1.00/02.0109 and ERDF BTU-AVOZ50520701. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.