Both fluorescence quantitative PCR and semi-quantitative RT-PCR can be used to detect gene expression, but the content of detection is somewhat different. Fluorescence quantitative PCR is to add a fluorescent dye (sybr green) that can specifically bind to double-stranded DNA or a fluorescent probe (taqman) that can bind to the target DNA fragment into the PCR reaction system. By monitoring the changes of fluorescence during PCR and comparing the changes of fluorescence of some housekeeping genes, the expression of the target gene can be reflected. The main valid data of fluorescence quantitative PCR is the time when the fluorescence signal increases to half of the maximum. Semi-quantitative RT-PCR indirectly reflects the abundance of the original template through the brightness of the electrophoresis band of the final product of RT-PCR. Because fluorescence quantitative PCR monitors the changes of PCR amplification in the whole process and can be relatively quantitative with the amplification curve of housekeeping genes, the accuracy of digital gene expression is higher than that of semi-quantitative RT-PCR. Semi-quantitative RT-PCR is the final product of monitoring, and when the gene expression level is high, the PCR system is likely to reach saturation in the middle and late stage of the reaction, so semi-quantitative RT-PCR can not accurately explain the problem for high-level expressed genes. However, if you want to publish a high-quality paper, some abnormal foreigners will not recognize the results of single fluorescence quantitative PCR, and they need the author to do a semi-quantitative RT-PCR as an auxiliary proof.

The primer design principles of semi-quantitative RT-PCR and fluorescence quantitative PCR are different, in other words, the primers of the two are not universal. Therefore, the annealing temperature is naturally different, and the specific difference depends on the primers actually used.