A quantitative real-time PCR method for tissue factor mRNA

Mälarstig, Anders; Tenno, Taavo; Jossan, Surinder; Åberg, Mikael; Siegbahn, Agneta

doi:10.1016/j.thromres.2003.11.001

Cited by 27 publications

(13 citation statements)

References 24 publications

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“…We have established that this technology gives highly reproducible results over 7 orders of magnitude, 19 which is consistent with previously published, conventional QRT-PCR data. 34 Recently, similar technology has been used to determine the prognosis of newly diagnosed, early-stage patients with breast cancer. The Oncotype DX assay (Genomic Health, Redwood City, CA) assesses the expression of a panel of 21 genes by QRT-PCR to identify patients with a high risk of recurrence and to identify patients who may benefit from certain types of chemotherapy.…”

Section: Discussionmentioning

confidence: 99%

Identification and Validation of Biomarkers of IgVH Mutation Status in Chronic Lymphocytic Leukemia Using Microfluidics Quantitative Real-Time Polymerase Chain Reaction Technology

Abruzzo

Barron

Anderson

et al. 2007

The Journal of Molecular Diagnostics

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To develop a model incorporating relevant prognostic biomarkers for untreated chronic lymphocytic leukemia patients, we re-analyzed the raw data from four published gene expression profiling studies. We selected 88 candidate biomarkers linked to immunoglobulin heavy-chain variable region gene (IgV H ) mutation status and produced a reliable and reproducible microfluidics quantitative real-time polymerase chain reaction array. We applied this array to a training set of 29 purified samples from previously untreated patients. In an unsupervised analysis, the samples clustered into two groups. Using a cutoff point of 2% homology to the germline IgV H sequence, one group contained all 14 IgV H -unmutated samples; the other contained all 15 mutated samples. We confirmed the differential expression of 37 of the candidate biomarkers using two-sample t-tests. Next, we constructed 16 different models to predict IgV H mutation status and evaluated their performance on an independent test set of 20 new samples. Nine models correctly classified 11 of 11 IgV H -mutated cases and eight of nine IgV H -unmutated cases, with some models using three to seven genes. Thus, we can classify cases with 95% accuracy based on the expression of as few as three genes. (J Mol Diagn 2007, 9:456 -555;

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Section: Discussionmentioning

confidence: 99%

Identification and Validation of Biomarkers of IgVH Mutation Status in Chronic Lymphocytic Leukemia Using Microfluidics Quantitative Real-Time Polymerase Chain Reaction Technology

Abruzzo

Barron

Anderson

et al. 2007

The Journal of Molecular Diagnostics

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“…Fluorescence emission was read by an Mx3000P (Stratagene; La Jolla, CA) functioning as a plate reader. First-strand cDNA synthesis using Omniscript (Qiagen) was performed on 2 g total RNA using a combination of oligo(dT) [12][13][14][15][16][17][18] and random hexamers (Amersham Pharmacia; Baie d'Urfé, QC, Canada) as primers. To ensure an RT performance of equal quality, all samples were reverse transcribed simultaneously using a single mastermix.…”

Section: Qrt-pcrmentioning

confidence: 99%

“…However, this option suffers from the drawback that it only normalizes a gene of interest against the average variation of many normalizing genes. While the ubiquitous normalizing gene may not exist, another more promising strategy is to identify a single normalizing gene for a single tissue type (27), pathology (12), or experimental design (5,16,18,28).…”

mentioning

confidence: 99%

Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon

Dydensborg¹,

Herring²,

Auclair³

et al. 2006

American Journal of Physiology-Gastrointestinal and Liver Physi

146

117

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As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include β-actin and glyceraldehyde-3-phosphate dehydrogenase. It has, however, become clear that these and other normalizing genes can display modulated patterns of expression across tissue types and during complex cellular processes such as cell differentiation and cancer progression. Our objective was to set the basis for identifying normalizing genes that displayed stable expression during enterocytic differentiation and between healthy tissue and adenocarcinomas of the human colon. We thus identified novel potential normalizing genes using previously generated cDNA microarray data and examined the alterations of expression of two of these genes as well as seven commonly used normalizing genes during the enterocytic differentiation process and between matched pairs of resection margins and primary carcinomas of the human colon using real-time RT-PCR. We found that ribosomal phosphoprotein P0 was particularly stable in all intestinal epithelial cell extracts, thereby representing a particularly robust housekeeping reference gene for the assessment of gene expression during the human enterocytic differentiation process. On the other hand, β-2-microglobulin generated the best score as a normalizing gene for comparing human colon primary carcinomas with their corresponding normal mucosa of the resection margin, although others were found to represent acceptable alternatives. In conclusion, we identified and characterized specific normalizing genes that should significantly improve quantitative mRNA studies related to both the differentiation process of the human intestinal epithelium and adenocarcinomas of the human colon. This approach should also be useful to validate normalizing genes in other intestinal contexts.

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“…When samples were genotyped, all of the individuals with the Ϫ1812 CC genotype and 5466 AA or AG genotypes were asked to donate heparin blood for isolation of peripheral blood mononuclear cells and subsequent measurement of TF mRNA and activity. 22,23 Please see http://atvb.ahajournals.org for details.…”

Section: Tf In Mononuclear Cellsmentioning

confidence: 99%

“…Quantities of TF mRNA were related to the expression of β-2-microglobulin, which has previously been found suitable as endogenous control in this kind of experimental setup (23). Differences in monocyte/lymphocyte ratio between donors were corrected for by using the cell count numbers.…”

Section: Tf Mrna In Mononuclear Cellsmentioning

confidence: 99%

Genetic Variations in the Tissue Factor Gene Are Associated With Clinical Outcome in Acute Coronary Syndrome and Expression Levels in Human Monocytes

Mälarstig

Tenno

Johnston

et al. 2005

ATVB

Self Cite

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Objective-Tissue factor (TF) has, among other factors, a prominent role in acute coronary syndrome (ACS). Our goal was to investigate whether single nucleotide polymorphisms (SNP) in the TF gene (F3) are associated with plasma TF, risk, and outcome in patients with ACS. Moreover, we wanted to investigate the impact of associated TF SNPs on mRNA production in human monocytes. Methods and Results-In 725 patients with ACS [Fragmin and Fast Revascularization during Instability in CoronaryArtery Disease II (FRISC-II) study] and 376 controls, 13 SNPs were genotyped and plasma TF measured. Thereafter, the 5466 AϾG and the Ϫ1812 CϾT were genotyped among all of the FRISC-II participants (nϭ3143) and assessed concerning clinical outcome. Associated SNPs were genotyped in 92 healthy blood donors for comparison of TF activity and TF mRNA expression. None of the SNPs were associated with patient/control status. The 5466 AϾG SNP was associated with cardiovascular death (odds ratio, 1.8; Pϭ0.025). The CG haplotype by Ϫ1812 CϾT and 5466 AϾG was associated with a 3-fold increased risk of death (PϽ0.001). TF mRNA and basal TF activity was significantly lower among 5466 AG carriers, whereas the increase in monocyte TF activity on lipopolysaccharide stimulation was significantly stronger (Pϭ0.04). Key Words: acute coronary syndrome Ⅲ tissue factor Ⅲ single nucleotide polymorphism Ⅲ outcome Ⅲ mRNA A cute coronary syndrome (ACS) is a multifactorial disease in which a multitude of lifestyle and genetic factors contribute to the development and outcome of the disease. 1 The genetic component of ACS seems to be relatively high, because family history is a strong risk factor, and heritability studies have shown a large genetic contribution to cardiac disease. 2,3 Single nucleotide polymorphisms (SNPs) are the most common genetic variations in the genome and may influence factors involved in atherosclerosis, plaque destabilization, and thrombosis, all of which are fundamental aspects of ACS etiology. 3 Variations in genes that encode for hemostatic factors are considered to be likely candidates for modifiers of the procoagulant phenotype, usually observed in patients with myocardial infarction (MI). 4 A procoagulant phenotype may have a substantial effect on ACS outcome, because a larger sized superimposed thrombus is directly related to the severity of myocardial ischemia. 5 A major player in this respect is tissue factor (TF), which rapidly initiates blood coagulation and is generally recognized as an important thrombogenic factor in atherosclerotic lesions. 6 TF is present as a membrane-bound molecule in different cell types in the vessel wall and within plaques, including endothelial cells, smooth muscle cells monocytes/macrophages, and foam cells. 7 Proteolytic cleavage of TF results in a soluble form of TF (plasma TF), which only contains the extracellular part of the protein and does not show significant procoagulant activity. 8 Moreover, the presence of an alternatively spliced form of TF has been reported recently. 9 Intravascula...

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A quantitative real-time PCR method for tissue factor mRNA

Cited by 27 publications

References 24 publications

Identification and Validation of Biomarkers of IgVH Mutation Status in Chronic Lymphocytic Leukemia Using Microfluidics Quantitative Real-Time Polymerase Chain Reaction Technology

Identification and Validation of Biomarkers of IgVH Mutation Status in Chronic Lymphocytic Leukemia Using Microfluidics Quantitative Real-Time Polymerase Chain Reaction Technology

Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon

Genetic Variations in the Tissue Factor Gene Are Associated With Clinical Outcome in Acute Coronary Syndrome and Expression Levels in Human Monocytes

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