Quantitative reverse transcription-PCR measurement of tissue factor mRNA in glioma

Background Initiation of the coagulation serine protease cascade in mammalian cells is mediated by tissue factor (TF), 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 of glioma. Methods 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/mug RNA. The relationship between C, and loo, starting concentration was linear (r(2) greater than or equal to 0.99). The mean expression of TF in healthy brain tissue was 6.2 x 10(3) copy/mug RNA. Overexpression of TF was found in 42 brain glioma samples, mean value is 2.9 x 10(6) copy/mug RNA. Conclusions TF niRNA transcript is expressed in glioma and the level of expression correlates with histologic 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. Index Entries: Glioma; tissue factor; mRNA.