1. Gdansk University of Physical Education and Sport, Faculty of Physical Education, Gdansk, Poland;
2. Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland;
3. Department of Physical Therapy, Ariel University, Ariel, Israel.
4. International Institute of Molecular and Cell Biology in Warsaw, Poland.
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.
Keywords: Brain-derived Neurotrophic Factor, Allele-specific detection, ddRT-PCR, Gene expression, Exercise metabolism, Reference gene