Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes.

For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet.

Thermolabile methylenetetrahydrofolate reductase, homocysteine, and cardiovascular disease risk: the European Concerted Action Project.

Background: Homozygotes for the thermolabile mutation (TT genotype) of the methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.

Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes).

Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction.

The C677T mutation in the methylenetetrahydrofolate reductase gene: a genetic risk factor for methotrexate-related elevation of liver enzymes in rheumatoid arthritis patients.

Objective: To study the possible relationship between the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene and the toxicity and efficacy of treatment with methotrexate (MTX) in patients with rheumatoid arthritis (RA).

Methods: Genotype analysis of the MTHFR gene was done in 236 patients who started MTX treatment with (n = 157) or without (n = 79) folic or folinic acid supplementation. Outcomes were parameters of efficacy of MTX treatment, patient withdrawal due to adverse events, discontinuation of MTX treatment because of elevated liver enzyme levels, and the total occurrence of elevated liver enzyme levels during the study.

Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons.

Background: Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses.

Methods: We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction.