In general, it can be divided into three phases: the fluorescence background signal phase, the fluorescence signal exponential amplification phase, and the plateau phase, which is shaped as a smooth S-shaped curve.
If there are many inflection points in the fluorescence background signal stage, the possible reason is that the system is not homogeneous or there are solid impurities; If you look up and then look down quickly after the downward probe, it may be because the amount of template in the system is too high, and it is recommended to dilute the template before use. If primer-dimers are present, the negative control will look up, which is difficult to avoid in real-time PCR; If the dissolution curve of the negative control shows the same peak as in the sample, there is contamination in the system configuration and the experimental results are not available.
There are three possibilities for the current doublet in the dissolution curve:
1. Primer peak, the primer peak is usually the first of the two peaks, and the way to eliminate it is to reduce the amount of primers in the system or redesign the primers;
2. When doing gene expression differences, it is easy to have DNA amplification peaks (only existing when primers cross introns), the reason for this is that there is DNA contamination during RNA extraction, which can be verified by electrophoresis, and the DNA in the RNA sample should be re-digested;
3. The amplification is non-specific, so the amplification conditions should be re-explored or the primers should be redesigned and verified.
Nucleic acid detection kits