sybr-green-i and Schizophrenia

Brain-derived neurotrophic factor (BDNF) plays a critical role in the growth, differentiation and survival of neurons in the CNS. Recent research has suggested that BDNF may be implicated in the etiology of mood disorders and schizophrenia, as well as in the therapeutic action of some drugs, such as antidepressants and antipsychotics. This study aimed to develop a simple, fast and accurate new method for detecting the Val66Met polymorphism of the BDNF gene in schizophrenia patients using melting-curve analysis and a DNA-specific dye, SYBR Green I. A group of 30 schizophrenia patients were analyzed to detect the BDNF Val66Met polymorphism (rs6265) using the new genotyping method based on the analysis of fluorescence melting curves of PCR products that were labeled with SYBR Green I. The genotype results were confirmed for all 30 samples using the specific BDNF TaqMan allele discrimination assay. This new method allows the analysis of both alleles in the same reaction tube using SYBR Green I, with no need for additional steps. The addition of a GC clamp makes this method universally applicable, since the melting temperature of one allele can be adjusted as necessary to give the distinctive separation of melting curves. Therefore, this new method is simple, fast and accurate for determining the presence of the BDNF Val66Met polymorphism. It may also be useful for the analysis of other SNPs in pharmacogenetic studies.

Topics: Alleles; Benzothiazoles; Brain-Derived Neurotrophic Factor; Diamines; DNA; Fluorescent Dyes; Genotype; Heterozygote; Homozygote; Hot Temperature; Humans; Methionine; Organic Chemicals; Polymerase Chain Reaction; Polymorphism, Genetic; Quinolines; Schizophrenia; Time Factors; Valine; White People

Quantitative polymerase chain reaction (Q-PCR) is now considered the "technique of choice" for validating gene expression changes identified with ribonucleic acid-based expression profiling technologies (especially micro- and macroarray techniques). The identification of altered gene expression profiles with microarrays is best viewed as the first step in the determination of potential disease-associated genes; however, the false-positive rate can be high, particularly with small sample sets and in view of the typically small differences observed in brain expression studies. Quantitative PCR is a rapid and highly sensitive technique for accurate quantification of microarray results; however, careful consideration of experimental design, quality of primer/probe design, internal standards, and normalization procedures are pivotal, particularly when the work involves postmortem tissue.

Topics: Apolipoprotein L1; Apolipoproteins; Benzothiazoles; Brain Chemistry; Diamines; Gene Expression; Humans; Lipoproteins, HDL; Myelin Basic Protein; Myelin Proteolipid Protein; Oligonucleotide Array Sequence Analysis; Organic Chemicals; Polymerase Chain Reaction; Postmortem Changes; Quinolines; Reproducibility of Results; RNA, Messenger; Schizophrenia; Sensitivity and Specificity; Statistics, Nonparametric