BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR - a Novel Protocol

Area of Science:

• Molecular Biology
• Neuroscience
• Genetics

Background:

• Brain-derived neurotrophic factor (BDNF) alterations are linked to neuropathological conditions.
• The Val66Met polymorphism in the human BDNF gene is a key area of investigation.
• No established methods exist for analyzing the impact of Val66Met on human BDNF function.

Purpose of the Study:

• To develop a quantitative reverse transcription PCR (qRT-PCR) protocol.
• To enable allele-specific expression evaluation of the Val66Met polymorphism in the BDNF gene.
• To provide a method for assessing BDNF functioning related to genetic variations.

Main Methods:

• RNA was extracted from muscle samples of healthy volunteers.
• A qRT-PCR protocol was developed for allele-specific BDNF expression detection.
• Validation was performed using Droplet Digital Polymerase Chain Reaction (ddPCR) for absolute quantification.

Main Results:

• The developed qRT-PCR protocol successfully detected allele-specific BDNF expression.
• ddPCR analysis confirmed differences in relative BDNF mRNA values.
• HPRT1 and B2M were identified as stable housekeeping genes in muscle under varying metabolic conditions, unlike GAPDH.

Conclusions:

• A novel qRT-PCR protocol was established for allele-specific detection of BDNF expression.
• The method accurately assesses changes in BDNF expression related to the Val66Met polymorphism.
• This provides a valuable tool for studying the functional impact of BDNF genetic variations.