RT-PCR generally refers to reverse transcription PCR.
The basic operation process is to reverse transcribe the extracted RNA, and then use the obtained cDNA as a template to design primers for amplification, which is a common method to detect gene expression.
Real-time fluorescence quantitative PCR is also called real-time PCR. Real-timePCR is to detect the progress of PCR in real time through the fluorescence signal during PCR amplification. In the exponential phase of PCR amplification, there is a linear relationship between the Ct value of the template and the initial copy number of the template, so it becomes a quantitative basis.
PCR principle:
Semi-conservative replication of DNA is an important way of biological evolution and passage. Double-stranded DNA can be denatured and unrolled into single strand under the action of many enzymes, and copied into the same two-molecule copy according to the principle of base complementary pairing with the participation of DNA polymerase.
It was found in the experiment that DNA can be denatured and melted at high temperature, and can be refolded into double chains when it is cooled. Therefore, the denaturation and renaturation of DNA can be controlled by temperature change, and DNA polymerase and dNTP can replicate specific genes in vitro by adding designed primers.
However, DNA polymerase will be inactivated at high temperature, so it is necessary to add new DNA polymerase every cycle, which is not only cumbersome to operate, but also expensive, which restricts the application and development of PCR technology.