The principle is different: real-time PCR detects the fluorescence excited by the fluorescent dye bound to DNA; ordinary PCR detects the presence of the target fragment by detecting the fluorescent stain in the inserted DNA.
On request: Fluorescence quantification has high requirements for amplified fragments, generally 100-300 bp; ordinary PCR can amplify long-point fragments. Fluorescence quantification allows quantitative analysis of products without electrophoresis, and ordinary PCR must be electrophoresed.
Results: Fluorescence quantification can achieve accurate quantification, but ordinary PCR does not. Fluorescence quantitative display can see the entire amplification process, you can see the amplification efficiency, dissolution temperature, directly obtained standard curve, etc., which are not possible with ordinary PCR.
There are two types of RT-PCR: real-time PCR (Real-Time PCR) and reverse transcription PCR (Reverse Transcription PCR), all of which belong to Q-PCR (Quantitative PCR), using RNA or cDNA as a template, and cDNA. Primers were designed to measure the expression of RNA levels.
Fluorescence quantitative PCR, plus fluorescence, must be done on quantitative PCR, the general fragment is shorter (80-120bp), there is a fixed procedure, and the difference in expression between different samples can be accurately calculated according to the control.
Reverse transcription PCR, no fluorescence, general PCR instrument, the number of cycles is between 15-25, the fragment can be selected 120-500bp, the expression difference can be displayed by running electrophoresis glue.
The commonly used method for Real-time PCR is SYBR Green. The primers of this method need to be based on RT-PCR:
1. Annealing temperature is the same as the internal reference;
2. The length of the segment should not exceed 200;
Real-time PCR primers can run RT-PCR, and vice versa.
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