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

Zhang H, Korenková V, Sjöback R, Švec D, Björkman J, Kruhøffer M, Verderio P, Pizzamiglio S, Ciniselli CM, Wyrich R, Oelmueller U, Kubista M, Lindahl T, Lönneborg A, Rian E - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: DiaGenic ASA, Oslo, Norway.

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.

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Time-course profile of EDTA down-regulation biomarkers in the validation study.1A: ATP2B4_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001); 1B: TNFRSF10C_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001). ΔCq = (Cqbiomarker – Cqmeanref) with Cqmeanref = mean of the Cq values of the 3 reference genes.
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pone-0111644-g001: Time-course profile of EDTA down-regulation biomarkers in the validation study.1A: ATP2B4_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001); 1B: TNFRSF10C_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001). ΔCq = (Cqbiomarker – Cqmeanref) with Cqmeanref = mean of the Cq values of the 3 reference genes.

Mentions: For EDTA down-regulation biomarkers, ATP2B4 and TNRFSF10C, and up-regulation biomarkers, LMNA, TNF, and FOSB, a significant change in expression was observed between T0 and T24 and T48 (Figure 1, 2). Furthermore, for each EDTA biomarker, we evaluated the relevance of the expression changes between T0 and the other two time points by computing the 95% simultaneous confidence interval (SCI) of the Log2 of the relative quantity (RQ). For biomarkers LMNA, FOSB and TNRFSF10C, the 95% SCIs showed significant changes from T0 in gene expression for both time points tested. For the other biomarkers, ATP2B4 and TNF, these changes were less pronounced, especially at T24 (Figure 3).


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

Zhang H, Korenková V, Sjöback R, Švec D, Björkman J, Kruhøffer M, Verderio P, Pizzamiglio S, Ciniselli CM, Wyrich R, Oelmueller U, Kubista M, Lindahl T, Lönneborg A, Rian E - PLoS ONE (2014)

Time-course profile of EDTA down-regulation biomarkers in the validation study.1A: ATP2B4_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001); 1B: TNFRSF10C_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001). ΔCq = (Cqbiomarker – Cqmeanref) with Cqmeanref = mean of the Cq values of the 3 reference genes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219744&req=5

pone-0111644-g001: Time-course profile of EDTA down-regulation biomarkers in the validation study.1A: ATP2B4_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001); 1B: TNFRSF10C_S (mixed model contrasts: T24 vs T0, p-value <0.0001; T48 vs T0, p-value <0.0001). ΔCq = (Cqbiomarker – Cqmeanref) with Cqmeanref = mean of the Cq values of the 3 reference genes.
Mentions: For EDTA down-regulation biomarkers, ATP2B4 and TNRFSF10C, and up-regulation biomarkers, LMNA, TNF, and FOSB, a significant change in expression was observed between T0 and T24 and T48 (Figure 1, 2). Furthermore, for each EDTA biomarker, we evaluated the relevance of the expression changes between T0 and the other two time points by computing the 95% simultaneous confidence interval (SCI) of the Log2 of the relative quantity (RQ). For biomarkers LMNA, FOSB and TNRFSF10C, the 95% SCIs showed significant changes from T0 in gene expression for both time points tested. For the other biomarkers, ATP2B4 and TNF, these changes were less pronounced, especially at T24 (Figure 3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: DiaGenic ASA, Oslo, Norway.

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.

Show MeSH