BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR - a Novel Protocol
| Autor: | Jay R. Hoffman, Eugene V. Gasanov, Gilmara Gomes de Assis |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Brain-derived neurotrophic factor
Messenger RNA biology Brain-derived Neurotrophic Factor General Medicine Molecular biology Housekeeping gene Real-time polymerase chain reaction Allele-specific detection ddRT-PCR Gene expression Exercise metabolism Reference gene Neurotrophic factors biology.protein Digital polymerase chain reaction Gene Glyceraldehyde 3-phosphate dehydrogenase Research Paper |
| Zdroj: | International Journal of Medical Sciences |
| ISSN: | 1449-1907 |
| Popis: | Background: Alteration in brain-derived neurotrophic factor (BDNF) production is a marker of neuropathological conditions, which has led to the investigation of Val66Met polymorphism occurring in the human BDNF gene (BDNF). Presently, there are no reported methods available for the analysis of Val66Met impact on human BDNF functioning. Purpose: To develop a qRT-PCR protocol for the allele-specific expression evaluation of the Val66Met polymorphism in BDNF. Methods: Using RNA extracted from muscle samples of 9 healthy volunteers (32.9 ± 10.3 y) at rest and following a maximal effort aerobic capacity exercise test, a protocol was developed for the detection of Val66/Met66 allele-specific BDNF expression in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) - relative to housekeeping genes - and validated by absolute quantification in Droplet Digital Polymerase Chain Reaction (ddPCR). Results: Differences in the relative values of BDNF mRNA were confirmed by ddPCR analysis. HPRT1 and B2M were the most stable genes expressed in muscle tissue among different metabolic conditions, while GAPDH revealed to be metabolic responsive. Conclusion: Our qRT-PCR protocol successfully determines the allele-specific detection and changes in BDNF expression regarding the Val66Met polymorphism. |
| Databáze: | OpenAIRE |
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