Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the intracellular folate metabolism which plays an important role in carcinogenesis through DNA methylation and nucleotide synthesis. The common MTHFR single nucleotide polymorphism C677T has been reported to be associated with reduced enzymatic activity. In order to investigate the influence of this polymorphism on the risk of chronic myeloid leukemia (CML), we performed a case-control study in a Serbian population of 52 patients with CML and 53 healthy control subjects. MTHFR C677T polymorphism genotyping was assessed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The results demonstrated no statistical difference in MTHFR 677 frequency distribution between patient and control groups. Our findings suggest that MTHFR 677 gene variants have no significant influence on the susceptibility to CML in a Serbian population.
The widespread use of gene expression analyses has been limited by the lack of critical evaluations of the methods used to extract nucleic acids from human tissues. For evaluating gene expression patterns in whole blood or leukocytes, the method of RNA isolation needs to be considered as a critical variable in the design of the experiments. Quantitative real-time PCR (qPCR) is widely used for the quantification of gene expression in today's clinical practice. Blood samples as a preferred RNA source for qPCR should be carefully handled and prepared in order not to inhibit gene expression analyses. The present study was designed to compare the frequently employed guanidine thiocyanate-phenol-chloroform-based method (TRI Reagent ® ) with two alternative RNA isolation methods (6100 PrepStation and QIAamp ® ) from whole blood or leukocytes for the purpose of gene expression analysis in chronic myeloid leukemia (CML) patients. Based on the results of this study, for the best combination of yield and RNA extraction purity, taking into account the necessary amount of the clinical sample and performance time, the protocol using phenol-based TRI Reagent ® for RNA extraction from leukocytes is suggested as the most suitable protocol for this specific gene expression analysis.
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