W:Brother, the reverse transcription is completed, and finally the last step – fluorescent quantitative PCR! Hey, brother, what is real-time quantitative PCR?
M:PCR you always know, right?
W:You know, PCR is the polymerase chain reaction, and it is a technology that increases DNA!
M:It’s almost… It is a technical method to amplify the target sequence to more than one million times through specific primers, but this only indicates the presence or absence of the target sequence, and cannot determine the amount of the initial template. Fluorescence quantitative PCR is a technical method developed to solve this problem. Real time PCR and quantity PCR (qPCR) generally refer to it.
W:The difference from ordinary PCR lies in fluorescence quantification?
M:Yes, that is, in the extension stage of each PCR cycle, the fluorescence signal intensity is detected, and the initial template amount can be calculated according to the change of the signal intensity.
W:It feels so complicated! Do I have to calculate this by myself?
M:This requires you to learn slowly, it is best to be able to understand the principle. Now you can focus on the CT value first, which is relatively intuitive experimental data, and its size corresponds to the size of the initial template amount. There are also graphs such as amplification curves and melting curves. They have the final say on whether your experiment is successful or not.
W:Oh, brother. Then I’m going to do the qPCR experiment!
M:Pay special attention not to add templates to the reagent area, otherwise your unused mix will be easily contaminated!
W:Got it!

Real Time PCR Easyᵀᴹ-SYBR Green I Kit

Higher efficiency, greater specificity
High sensitivity, good compatibility