If we want to study gene expression we must do a reverse transcription to convert mRNA from our total RNA samples into cDNA before performing qPCR. By doing this we’ll be able to amplify expressing genes, and as our cDNA templates lacks introns, we could design primers that only amplifies cDNA, avoiding this way the amplification of gDNA contaminants or if it is not possible, design primers separated by at least one intron to discriminate between cDNA-amplified products from gDNA-amplified ones.
This protocol is optimized to fit our needs from the original manufacturer’s protocol. We are using a commercial from Bio-rad (iScript™ cDNA Synthesis Kit). Manufacturer’s full protocol is available on their webpage.