Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

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.

Effects of post-mortem and physical degradation on RNA integrity and quality

“…Accurate quantification of gene expression with methods such as RT-qPCR, microarray profiling and next-generation sequencing requires integral RNA of high quality. It is well established that the pre-analytical steps in molecular analysis, comprising sample collection, transportation, storage, and extraction, may have profound effect on the RNA and, via reverse transcription, the cDNA quality and introduce substantial technical bias [2] , [3] , [4] . It is therefore critical to use highly optimized and validated pre-analytics, but also to test the quality and integrity of the extracted RNA.…”

“…The post-mortem degradation of nucleic acids in biological samples has proven useful in forensic pathology, where the time of death can be estimated [1] . In diagnostic samples post-mortem nucleic acid degradation is only a nuisance that should be controlled and kept to a minimum [2] , [3] , [4] . Usually the confounding processing (technical) variation introduced by DNA degradation is small and can be ignored.…”

Preanalytical Variables Affecting the Integrity of Human Biospecimens in Biobanking

“…Preanalytical storage factors affecting analyte quantity, quality, or gene expression are temperature, storage time, concentration of RNA, and repeated thaws (53,119,120 ). As for RNA, new technologies for dry storage at room temperature have been developed (121)(122)(123).…”

Stabilization of circulating thyroglobulin mRNA transcripts in patients treated for differentiated thyroid carcinoma

“…Such collection tubes include the PAXgene and Tempus Blood RNA tubes, which appear to stabilize gene expression in blood when compared with common collection tubes such as EDTA tubes. [24][25][26][27] Apart from being a prerequisite for clinical application of RT-PCR-based TG assays, control of preanalytical variables is of utmost importance to obtain reliable analytical results, when testing the ability of RT-PCRbased TG assays to predict residual, recurrence or metastatic disease in follow-up of DTC patients after treatment. Although important only Eszlinger et al have examined the influence of preanalytical variables on blood TG mRNA levels.…”