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


METHODS
Blood was collected from healthy volunteers in K(3) 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 K(3) EDTA tubes. The PAXgene tubes preserved high molecular mass rRNA better than the other tubes.


CONCLUSIONS
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 microg extracted RNA. A time frame up to 24 h until sample processing is acceptable for TF and VEGF mRNA.
Department of Clinical Chemistry, Orebro University Hospital and Division of Biomedicine, Orebro University, S-70185 Orebro, Sweden.
Helena S
HS
Isaksson
Torbjörn K
TK
Nilsson
D018076:DNA, Complementary; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013925:Thromboplastin / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D013925:Thromboplastin