Detection of circulating tumour cells in blood by quantitative real-time RT-PCR:: effect of pre-analytical time

Background: We and others have recently explored the use of quantitative real-time RT-PCR analysis for the detection of circulating tumour cells in blood of patients with breast cancer (BC). One major problem in these experiments is the in vitro instability of the cellular RNA. The copy number of mRNA can change during storage and transport at room temperature and this may hamper accurate quantitative measurements of specific transcripts, especially when working with small numbers of target mRNAs. Methods: Peripheral blood samples were obtained from two healthy volunteers and 13 patients with BC. Blood was stored at room temperature for 0, 1, 2, 4, 6, 24, 48 and 72 h. The potential alteration of gene expression for six target genes was investigated by quantitative real-time RT-PCR. Results: For beta-actin, GAPDH, cytokeratin-19 (CK-19) and HER2, a significant decrease in expression level occurs after 4 h (CK-19 and HER2), 6 h (beta-actin) or 24 h (GAPDH). Mammaglobin expression was only measurable in two samples and seems to be stable for at least 6 h. For vascular endothelial growth factor (VEGF), a statistically significant increase in expression level is observed in samples processed 24 h after collection. Conclusions: Most transcripts were reduced in samples that were stored overnight at room temperature compared with fresh samples, but upregulation of transcripts, probably as an active response to cellular stress, also occurs when blood is removed from its in vivo environment and stored ex vivo. Optimally, blood samples and RNA should be processed or stabilised within 3 h after collection to avoid interference of the in vivo gene expression signature by ex vivo stress responses.