Real-Time PCR (qPCR), namely the real-time fluorescent quantitative nucleic acid amplification detection system, is a method and technology that uses fluorescent dyes to detect the total amount of products after each PCR cycle, and is a derivative reaction of conventional PCR. It is mainly to monitor the changes in the amount of amplified products in each cycle of the PCR amplification reaction in real time through the changes of fluorescent signals, and to quantitatively analyze the starting template through the relationship between the ct value and the standard curve.
 
Common problems and solutions in the use of Real-Time PCR (qPCR) are as follows:
I. The repeatability is poor
The reasons for the poor repeatability of qPCR experiments are as follows:
1. The inaccurate pipette leads to inaccurate sample addition. In this case, we can change another pipette with better performance, expand the reaction volume, dilute the template at high times, and add to the reaction system in a large volume.
2. The temperature of the quantitative PCR instrument is different in different positions. This requires regular calibration of it.
3. If the qPCR master mix is not evenly mixed, mix it thoroughly before use.
Note: The amplification curve with ideal repeatability STD<0.2
 
II. The melting curve is a non-single peak
The possible reasons for this problem are as follows:
1. For non-specific amplification, new primers can be designed according to design principles, and primer annealing temperature can be optimized by gradient PCR.
2. A primer dimer appears, and the primer is a non-specific primer.
The peak value of the primer dimer in the melting curve caused by unreasonable primer design is generally located at about 75°C. If the peak is distinct, please optimize according to the following aspects:
(1) Optimize the amplification conditions and set the gradient Tm to find the best Tm value.
(2) The primer concentration is too high, appropriately reduce it.
(3) The primer dimer can be confirmed by agarose gel electrophoresis.
3. The template has genome contamination
In this case, just need to prepare the cDNA template again.
 
III. Ct value appears too late
1. The PCR product is too long. The PCR product should not be too long, usually between 100-50bp.
2. The cDNA template is degraded to re-prepare a new template and repeat the experiment.
3. The amplification efficiency is extremely low. Optimize the reaction conditions, try the three-step amplification procedure, or redesign the primers.
4. There is a PCR reaction inhibitor in the reaction system. Increase the template dilution factor or re-prepare the template to repeat the experiment.
5. cDNA template concentration is low. Reduce dilution and repeat the experiment.
 
IV. The Ct value of the internal reference gene is normal, but the Ct value of the target gene appears late
The Ct value of the internal reference gene is normal, indicating that the reagents and operation steps are correct. The possible reasons why the Ct value of the target gene appears late are as follows:
1. The expression of the target gene is low. In this case，re-enrich RNA.
2. The amplification efficiency of target gene primers is low:
(1) Dilute the template stepwise to determine the amplification efficiency of primers.
(2) Lower the annealing temperature.
(3) Redesign primers.