Why was the reverse transcriptase used?
Reverse transcriptase was used to convert RNA to complementary DNA (cDNA) which was used as a template in the qRT-PCR reaction for detection of gene expression products. Brilliant II SYBR Green PCR Master Mix provided all the components and reagents needed for the reaction. Careful primer design was critical to prevent undesired primer complementarity which could affect the accuracy of results. DNA was amplified using the same three steps as standard PCR: denaturation; annealing; and extension at relevant temperatures and SYBR green dye was used as a fluorescent label (due to its high affinity for dsDNA) enabling collection of data as the PCR progressed and collection and quantitation of gene transcripts. The amount of fluorescence measured was proportional to the PCR product, and therefore, the DNA could be quantified in real-time. The rate at which fluorescence develops Is dependent on the number of DNA molecules in the initial sample- the more DNA molecules present, the faster the fluorescence will increase.