Quantitative Reverse Transcription-PCR Measurement of Tissue Factor mBNA in Glioma

摘要

Background Inititation of the coagulation serine protease cascade in mammalian cells is mediated by tissue factor (FT), which is a cell surface receptor and cofactor for coagulation factor VII (FVII) and its activated form FVII (FVIIa). Increasing evidence suggests that TF is expressed in a wide range of cancer cells and plays important roles in cancer progression and metastasis. In this study, we investigated the association between the expression level of TF transcript and histologic features fo glioma.Medthods RNA was extracted from normal brain tissues and glioma tissues. We developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify TF gene expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the mRNA level in human glioma.Results The dynamic range of the assay was 10~3-10~8 copy/#mu#g RNA. The relationship between C_t and log starting concentration was linear (#gamma#>=0.99). The mean expression of TF in healthy brain tissue was 6.2 X 10~3 copy/#mu#g RNA. Overexpression of TF was found in 42 brain glioma samples, mean value is 2.9 X 10~6 copy/#mu#g RNA.Conclusions TF mRNA transcript is expressed in glioma and the level of expression correlates with hisologic grade of malignancy. This new simple, rapid, semiautomated assay is a major alternative to Northern blot and competitive quantitative PCR for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and support future TF-based clinical applications.