A quantitative real-time PCR method for tissue factor mRNA.

Anders Mälarstig; Taavo Tenno; Surinder Jossan; Mikael Aberg; Agneta Siegbahn

doi:10.1016/j.thromres.2003.11.001

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A quantitative real-time PCR method for tissue factor mRNA.

Anders Mälarstig·Taavo Tenno·Surinder Jossan·Mikael Aberg·Agneta Siegbahn

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Abstract

BACKGROUND: Tissue factor (TF) is primarily known for its function to initiate blood coagulation. The range of in vivo expression of TF is wide and requires a dynamic assay for monitoring. A general method for TF mRNA quantitation that is dynamic, sensitive and applicable to a variety of experimental systems or clinical situations is therefore desirable. OBJECTIVES: To develop a method for sensitive and dynamic quantitation of TF mRNA in human blood cells. METHODS: TF mRNA expression was analysed and evaluated in monocyte isolations, in whole blood (healthy volunteers and patients scheduled for percutaneous coronary intervention, PCI) and in a panel of human cell lines. RNA was extracted, reverse transcribed and subjected to real-time PCR amplification, according to the TaqMan technology. A TF plasmid was constructed as calibrator of the assay. Two housekeeping genes used as endogenous controls for cDNA quality and integrity were evaluated. RESULTS: The assay was linear by seven orders of magnitude and detected down to 10(2) copies of the TF plasmid. The coefficient of variation was 4% intra-assay and 28% between the assays when using beta2MG as endogenous control. The beta-actin gene expression was induced by treatment with lipopolysaccharide (LPS) in blood leukocytes and could not be used as an endogenous control. However, beta2MG showed only minor variations upon treatment with LPS. The TF mRNA and antigen expression, measured in a Western blot, correlated well (R(2)=0.903) in a panel of 11 human cell lines. CONCLUSIONS: We have established a method for sensitive and dynamic quantitation of TF mRNA in experimental systems and for clinical situations.

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Base Sequence Breast Neoplasms Calibration Cell Line, Tumor Cells, Cultured DNA Primers DNA Probes Female Humans Male Plasmids Polymerase Chain Reaction Prostatic Neoplasms RNA RNA, Messenger Sensitivity and Specificity Thromboplastin Journal Article Research Support, Non-U.S. Gov't

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Anders Mälarstig, Taavo Tenno, Surinder Jossan, Mikael Aberg, Agneta Siegbahn. 2003. A quantitative real-time PCR method for tissue factor mRNA.. https://doi.org/10.1016/j.thromres.2003.11.001

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