To determine protein expression levels in the brain using qRT-PCR, you would need to follow these general steps:
Sample preparation: Collect brain tissue samples from the region of interest and extract the RNA.
Reverse transcription: Use reverse transcriptase to convert the RNA into cDNA, which is the template for qRT-PCR.
Primer design: Design specific primers for the gene of interest that will be used to amplify a specific region of the cDNA during qRT-PCR.
PCR amplification: Amplify the cDNA using the specific primers in a real-time PCR machine. The machine will measure the fluorescence of the PCR product in real-time, allowing you to track the amplification of the target gene.
Data analysis: Analyze the data generated by the real-time PCR machine to determine the relative expression levels of the gene of interest. This is typically done by normalizing the target gene expression to a reference gene (housekeeping gene) that is known to be stably expressed across different samples.
Validation: Validate the results by comparing the expression levels of the protein of interest with other methods such as Western blot or Immunohistochemistry