What protocol is best for determining the protein expression level in the brain using quantitative RT-PCR?
Brain tissue preservation suitable for the protocol and how to analyse
Rodolfo Bolaños Barrera
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
To determine protein expression levels in the brain using qRT-PCR, you would need to follow these general steps:
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