How do i observe gene level through using real time quantitative pcr?
It helps to know what PCR does. Basically, it takes a small amount of DNA (usually the whole genome) and amplifies just the portion you want. It uses primers for both strands of the double helix to isolate the DNA section of interest. Genes are expressed when mRNA is formed – a process called transcription. Translation is needed to turn that mRNA into protein, but PCR doesn’t deal with proteins. In order to apply PCR, you need to use than mRNA. However, PCR requires a double strand. Solution: reverse transcriptase. This makes mRNA into DNA so that it can be used as a template in PCR. So now you’ve got PCR going. The next step is to sample how much of your target gene you get after predetermined rounds of amplification. Run samples of your rounds on a single gel so you can see differences in band strength. There are computer programs that will analyze the width/strength of those bands so you can get quantitative analysis of that PCR.
