Week 7: RT-QPCR
Welcome back to my blog! In the coming weeks, I plan to do use Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). How RT-qPCR differs from normal Polymerase Chain Reaction is that RT-qPCR uses reverse transcription with mRNA samples, instead of straight up DNA in PCR. The importance of using mRNA lies in the fact that mRNA directly shows the gene expression of specific genes in different responses, like the inflammatory response in my project, whereas DNA just has all the genes, which is the same for each cell. In RT-qPCR, the mRNA is reverse transcribed using an enzyme called reverse transcriptase to add another complementary strand to create a cDNA copy, which is then amplified to make billions of cDNA copies. These cDNA copies can then translated into a graph which is then analyzed to identify specific markers of inflammation.
In this research, Chinese Hamster Ovary (CHO) cells will first be induced to produce lots of inflammatory proteins by adding inflammatory cytokines such as IL-6. Producing many inflammatory proteins will require differential gene expression of certain genes either promoting inflammation or inhibiting anti-inflammatory factors. Thus, to identify these genes and other markers of inflammation, it is important to isolate the mRNA of the cells and go through RT-qPCR to amplify the genetic information to see the certain genes involved in inflammation.