Part 1 Abnormal amplification curves Normal amplification curves are generally S-shaped, with Ct values preferably between 20 and 30. Abnormal amplification curves include large Ct values, no plateau period, and decreasing plateau period. 1. Large Ct value (e.g. Ct value >30)
(1) Low template amount or low abundance of gene expression, it is recommended to increase the template amount to see if the Ct value can be reduced by a corresponding factor.
(2) The qPCR reaction conditions are not suitable or the primers are not properly designed, resulting in low amplification efficiency, it is recommended to confirm the amplification efficiency through the standard curve.
(3) The amplification product is too long, it is recommended to use the three-step procedure to amplify or optimize the primers, and the length of the amplification product should not exceed 300bp.
(4) Inhibitors may exist in the system to affect the enzyme activity, it is recommended to dilute the template in gradient or re-prepare the template with higher purity.
2. The amplification curve fails to reach the plateau period The gene abundance is low and the number of cycles is small, it is recommended to increase the number of cycles or choose products suitable for the quantification of low abundance genes.
3. Decrease in the plateau period of amplification curve It may be due to improper setting of the baseline range, which is usually caused by the amount of template is too high. It is recommended to reduce the end point value of the baseline (generally recommended to set the Ct value -4), after adjustment, see the following figure.
4. Jagged amplification curve plateau period
(1) The purity of RNA is low, it is recommended to increase the dilution of template in gradient to see the optimization effect or re-prepare high-purity RNA to re-experiment.
(2) The instrument has not been calibrated for a long time, it is recommended to calibrate and maintain the instrument regularly.
5. The amplification curve is messy and irregular. It may be that the concentration of ROX does not match the model, it is recommended to adjust the concentration of ROX, after adjustment, see the figure below. Note: You can change the reference dye setting from ROX to NONE on the instrument, cancel the correction function of ROX, and check whether the amplification curve returns to normal to determine whether it is caused by ROX.
6. There is melting curve but no amplification curve. It may be that the amplification program is set incorrectly and the fluorescence signal is not collected, so it is recommended to re-experiment and increase the collection of fluorescence signal in the extension phase of the amplification program.
7. the amplification curve has upward or downward spikes It may be a fault of the instrument, it is recommended to repair the instrument.
8. Negative control has amplification
(1) NTC has amplification, there may be the following two cases: ① Ct>35, melting curve Tm value <80 ℃ (general normal qPCR products, size between 100-300bp, melting curve Tm mostly above 80 ℃), may be the result of primer dimer, can be further optimization of primers. ② There is a Ct value and <35, indicating that the reaction system is contaminated, can be gradually excluded from the source of contamination.
(2) NRC has amplification NTC is normal, NRC has Ct value, may be RNA contains gDNA contamination. It is recommended to digest with DNase I or use a reverse transcription kit containing gDNA removal (e.g. Cat NO.11141ES). [Note] NTC is a negative control reaction in which H2O is substituted for the template; NRC is a negative control reaction in which non-reverse transcribed RNA is used as the template.
9. Poor repeatability of duplicate wells
1) The addition error causes, it is recommended that the pipette is calibrated regularly, in addition, it can be optimized by expanding the reaction system or preparing a good premix in advance.
2) Low template volume, it is recommended to increase the template volume, so that the Ct value falls between 15-30.
(3) The instrument has not been calibrated for a long time, it is recommended to calibrate and maintain the instrument regularly.
Part2 Melting curve abnormality Melting curve is often used to determine the specificity of qPCR results, the ideal melting curve is a single peak, the abnormal melting curve may appear double peaks, messy and so on, we will analyze the following one by one.
1. Melting curve with double peaks
1）Double peak, spurious peak Tm before 80℃ ①There may be primer dimer, it is recommended to optimize by reducing the primer concentration or redesigning the primer. It is recommended to optimize by reducing the primer concentration or redesigning the primer. ② It is possible that the amount of template is too low, which contributes to the formation of primer dimer, and it is recommended to increase the template amount.
(2) Double peaks, miscellaneous peaks Tm after 80 ℃ ① primer specificity is too poor resulting in non-specific product amplification, it is recommended that Blast check primer specificity or redesign primers. ② gDNA contamination, can be confirmed by NRC. If the NRC has Ct value, the template can be re-prepared.
2. melting curve single peak but not sharp There may be non-specific products of similar size, it is recommended to confirm by high resolution agarose electrophoresis.
3. melting curve single peak but Tm value before 80 ℃ Presumably no template, only the primer dimer amplification. Confirm the product by high resolution agarose electrophoresis or repeat the experiment.
4. Melting curve peak shape is messy
1) Contamination of the reaction system, confirm the contamination by combining the NTC and NRC results, and suggest eliminating the contamination one by one from water, primer, enzyme and environment.
(2) Reagent failure due to exposure to bright light or high temperature, suggest using new reagents for comparison.
(3) The instrument has not been calibrated for a long time, it is recommended to calibrate and maintain the instrument regularly.
4) Consumables do not match with the instrument, confirm the requirements of the corresponding instrument for consumables.
5. Amplification curve but no melting curve It may be that the melting program is wrongly set, and the fluorescence signal is not collected, so it is recommended to re-experiment and increase the signal collection in the melting curve stage.
6. When two reagents are compared in parallel, the Tm value of the same product is different. It may be that different reagents have different composition or content of pro-dissolution chain, which leads to different Tm values of the same product at the dissolution temperature.
7. The front end of the melting curve may be a mismatch between the concentration of ROX and the model, it is recommended to cancel the ROX calibration to see if the melting curve is normal, adjusted as shown below. Note: You can change the reference dye setting from ROX to NONE on the instrument, cancel the correction function of ROX, and check whether the melting curve is back to normal, then you can decide whether it is caused by ROX.