Preanalytical aspects of quantitative TaqMan real-time RT-PCR: Applications for TF and VEGF mRNA quantification

Objectives:The present paper focuses on preanalytical aspects of tissue factor (TF) and vascular endothelial growth factor (VEGF) mRNA quantification: the choice of blood collection tubes and defining the time frame allowed before processing the sample.Design and methods:Blood was collected from healthy volunteers in K3 EDTA tubes, CPT™, endotoxin-free EndoTube™ tubes and in PAXgene™ tubes. Total RNA concentration was determined by absorbance readings at 260 nm with a GeneQuantII UV spectrophotometer. RNA quantity and quality were also determined by the Lab on a Chip technique (Agilent 2100 Bioanalyzer). Real-time RT-PCR assays were performed by the TaqMan technology.Results:The more expensive PAXgene and CPT tubes and the Endo tubes did not give superior results from those obtained in inexpensive routine K3 EDTA tubes. The PAXgene tubes preserved high molecular mass rRNA better than the other tubes.Conclusion:Both the PAXgene™ system and routine EDTA tubes are suitable for clinical purposes aimed at quantitation of mRNA for TF and VEGF. PAXgene™ yielded rRNA that was less degraded but had lower mRNA per μg extracted RNA. A time frame up to 24 h until sample processing is acceptable for TF and VEGF mRNA.