Quantitative Tissue Factor Gene Expression Analysis in Whole Blood: Development and Evaluation of a Real-Time PCR Platform

Up-regulation of tissue factor (TF) expression in circulating blood cells, especially monocytes, plays a key role in the pathogenesis of various thromboembolic diseases (1)(2)(3)(4). Measurement of TF expression in monocytes therefore might be helpful in the diagnosis of a hypercoagulable state (5)(6)(7). Monocytic TF expression can be measured on both the protein and RNA levels. Testing on the RNA level seems to be more appropriate than antigen testing to detect a procoagulant state because monocytes that already express functionally active TF on their membrane surfaces are rapidly cleared from the circulation (8).The aim of the present study was to develop a protocol for quantitative determination of TF mRNA transcripts in monocytes and other circulating blood cells. The method should be rapid, robust, and applicable in whole blood without the need for cell isolation. We developed a one-step quantitative reverse transcription (qRT)-PCR assay based on the real-time TaqMan® technology and evaluated the preanalytical conditions required for the use of TF mRNA measurements on a routine clinical basis as well as several normalization strategies, including normalization based on CD14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene expression. Finally, we measured baseline TF expression in healthy individuals and in thrombophilic patients.RNA calibrators for quantification were prepared by in vitro transcription of DNA hybrids that combined the T7 promoter sequence with the following sequences of TF, CD14, or GAPDH cDNA: for TF, bp 265–848 (GenBank accession no. M16553); for CD14, bp 18–564 (GenBank accession no. M86511); and for GAPDH, bp 8–525 (GenBank accession no. M33197). Generated RNAs were treated with DNase I (Roche) to remove the added DNA templates and purified by use of the RNeasy Mini Kit (Qiagen). RNA was quantified by photometric measurement ( A 260 reading of 1 = 44 …