Real-Time PCR (qPCR) has developed into an important tool in molecular biology research because of its sensitivity, specificity, accuracy, and ease of use.
 
Common problems and solutions during the use of Real-Time PCR (qPCR) are as follows:
V. Abnormal amplification curve, such as "S" curve
1. The amplification curve caused by the reaction system is not smooth---amplification efficiency deviation (too high or too low)
(1) The amplification efficiency is too high
Non-specific amplification or primer dimers: too high a template concentration in the reaction system and poor template nucleic acid quality may cause inhibition of the PCR reaction. In absolute quantification, the amplification efficiency is greater than 110%.
Solutions:
①Remove the reaction well with the highest template concentration and re-analyze the standard curve. If the efficiency returns to below 110%, the analysis is good;
② Make a standard curve for the target gene, generally use the plasmid that clones the gene to do a gradient dilution, or use the PCR product to do a gradient dilution, and then do quantitative amplification, and evaluate the reaction efficiency through the curve. Absolute quantitative and effective amplification efficiency is 90%-110%;
③ Re-purify the template to remove potential inhibitors in the template. Remember to extend the drying time to remove ethanol during ethanol precipitation, or use another purification column and add washing solution to remove chaotropic salts from the purified silica gel.
(2) The amplification efficiency is too low
It is mainly manifested in the improper reagent concentration (mainly primers, magnesium ions and Taq DNA polymerase), especially in multiple experiments where the difference in primer pair Tm exceeds 5°C and the thermal cycling conditions are not suitable, the competition of various homologous substances in the test tube can cause low reaction efficiency. Absolute quantitative performance: amplification efficiency is <90%.
Solutions:
Adjust and optimize the experimental system after eliminating the above factors one by one.
(3) Abnormal amplification curve
For example, a sudden drop in individual amplification curves: there are air bubbles in the reaction tube, and because the bubbles burst after the temperature rises, the fluorescence value detected by the instrument drops suddenly.
Solutions:
Before proceeding with the amplification reaction, carefully check whether there are air bubbles remaining in the reaction tube.
2. Abnormal curve caused by improper instrument setting
Improper setting of the baseline, such as broken or slipped amplification curve (the end point of the baseline is greater than the Ct value). Reduce the baseline end point (Ct value-4), reanalyze the data;
(1) Improper setting of baseline range and threshold
Both the baseline range and threshold are artificially set parameters. The two are generally automatically defaulted to appropriate values by the instrument. When evaluating multiple kits or chemicals in the same program, improper threshold settings often result in differences in the amplification curves of different groups of data: the threshold automatically selected by the software is more suitable for the higher curve of the platform, which will cause deviations in the Ct value in the data group, because its optimal threshold is lower than this value. Therefore, it is necessary to conduct independent research on each data group so that the best threshold can be selected according to the specific situation;
(2) Rox improperly added
It shows that the amplification curve is jagged and discontinuous, and the reference dye needs to be corrected.
 
VI. Poor linear relationship of standard curve in absolute quantification
1. Sampling error
Increase the template dilution factor and enlarge the sample volume
2. Degradation of standard products
Prepare the standard products again and repeat the experiment
3. The template concentration is too high
Increase the template dilution factor