Q1: What are the things to consider when designing primers for qPCR?

A1: (1) The recommended size of amplification products is 80 – 200 bp. The smaller the size, the higher the amplification efficiency. The product size should be over 80 bp to distinguish from primer dimers, but if the size is too large, the amplification efficiency will be decreased;

(2) Avoid GC-rich or AT-rich regions at the 3' end;

(3) The last base should be G or C instead of T;

(4) The difference in Tm values of the forward and reverse primers should be no more than 1°C;

(5) The GC content should be 40%–60%;

(6) For probe-based qPCR, the Tm value of probes should be 8°C–10°C higher than that of primers.

Q2: Should the cDNA template be diluted? What's the dilution factor for qPCR?

A2: There is no reference dilution factor. In general, the Ct value increases by 3.3 with each 10-fold dilute solution of cDNA. Plan dilutions based on this rule. Perform qPCR using cDNA stock solution, 10-fold dilute solution, and 100-fold dilute solution as templates, and choose a dilution factor that gives a Ct value within the range of 18–28 or 15–33 based on the above rule. Alternatively, refer to the dilution factor of the previously used kits.

Note: When using the cDNA stock solution for testing, the input amount should not exceed 1/10 of the qPCR system, as the cDNA contains many components that inhibit qPCR and may hamper qPCR at large volumes.

Q3: The shapes of amplification curves are abnormal.

A3: (1) The amplification curve is unsmooth: The signal is too weak and the unsmooth curve is generated after system calibration. Repeat the experiment after increasing the template concentration.

(2) The amplification curve breaks off or declines: Because of the high template concentration, the baseline end cycle is greater than the Ct value. Decrease the baseline end cycle (Ct value - 4) and re-analyze the data.

(3) Sharp drops in certain amplification curves: Residual bubbles in the reaction tube burst with rising temperature, leading to a sudden drop in fluorescence intensity detected by the instrument. Inspect reaction tubes carefully for residual bubbles before the assay.

(4) The amplification curve is jagged and discontinuous: ROX is not added properly. Calibrate the reference dye.

Q4: The standard curve shows an amplification efficiency lower than 90% or higher than 120% and a poor linear relationship.

A4: (1) Sample loading error. Increase the template dilution factor and increase the sample volume. Use different dilution gradients to derive more accurate concentrations;

(2) The standard has degraded. Prepare a new standard and repeat the experiment;

(3) The template concentration is too high and may inhibit the reaction. Increase the template dilution factor;

(4) The primers have poor amplification specificity. Redesign primers and repeat the experiment.

Q5: No amplification curve appears at the end of the reaction.

A5: (1) Insufficient reaction cycles: In general, the number of cycles is set to 40. However, too many cycles will increase background signal, reducing data credibility.

(2) Check whether the signal acquisition step has been set up in the program: Two-step qPCR generally sets up signal acquisition at the annealing and extension phases; three-step qPCR sets up signal acquisition at the 72°C extension phase.

(3) Inappropriate primers: Redesign primers; check whether primers have degraded: The integrity of primers stored for a long time should be first tested by PAGE electrophoresis to rule out potential degradation.

(4) Too low template concentrations: Reduce the dilution factor and repeat the experiment. For samples of unknown concentrations, test the highest concentration first.

(5) Template degradation: Prepare new templates and repeat the experiment.

Q6: Criteria for validity of Ct values

A6: (1) Single peak in the melt curve (dye-based)

(2) In the exponential phase of the amplification curve, the standard deviation (STD) of Ct values is < 0.2 among replicate wells.

(3) Appropriate threshold

(4) The experiment with the no-template control (NTC) indicates no or negligible aerosol contamination.

(5) The experiment with the no reverse transcriptase control (NRT) indicates no or negligible contamination by residual genomic DNAs.

(6) The amplification efficiency (e) meets approximation criteria and is 95%–105% or 90%–120%; the correlation coefficient (R2) of the standard curve is over 0.98.

Q7: The Ct values are too large.

A7: (1) The amplification efficiency is too low. Optimize reaction conditions by using the three-step qPCR program or redesigning synthetic primers.

(2) The template concentration is too low. Reduce the dilution factor and repeat the experiment. For samples of unknown concentrations, test the highest concentration first.

(3) Template degradation. Prepare new templates and repeat the experiment.

(4) The PCR product is too long. The recommended PCR product size is 80 bp–150 bp.

(5) The system contains PCR inhibitors. Inhibitors are generally introduced by the template. Increase the template dilution factor or prepare a new template, and repeat the experiment.

Q8: If NTC has a Ct value, can the Ct value of the target gene still be used?

A8: NTC amplification generally involves two scenarios:

(1) The peak pattern of the melt curve does not overlap between the NTC and the target gene. The NTC generally has a lower Tm than the target gene. In this case, the Ct value of NTC is caused by primer dimers, and will not affect the acquisition of the Ct value of the target gene.

(2) The peak pattern of the melt curve overlaps between the NTC and the target gene. The NTC has the same Tm as the target gene.

This suggests that the system has been contaminated by aerosols. In this case, can the Ct value of the target gene still be used?

This needs to be determined by calculating the difference in Ct value (△Ct) between the target gene and NTC. △Ct ≥ 5/3 indicates that aerosol contamination has minimal influence on the system and is negligible. △Ct ＜ 5/3 indicates severe contamination; in this case, the Ct value of the target gene cannot be used.

Q9: Significant amplification occurs in the NRT group.

A9: Significant amplification of the NRT suggests genomic DNA contamination. The usability of experiment data can be determined from △Ct between the test group and the NRT group. △Ct > 5 indicates that the deviation induced by gDNA contamination is less than 5% and negligible. △Ct < 3 or △Ct ≈ 0 indicates that the products in the test group are mostly amplified from gDNAs, which nulls the qPCR quantification. In this case, perform RNA extraction using an RNA extraction kit with a genome removal module, or include the genome removal step during reverse transcription.

Q10: The melt curve has multiple peaks.

A10: (1) Poor primer design: Design and synthesize new primers based on the primer design principle;

(2) The primer concentration is too high: Reduce the primer concentration appropriately;

(3) The cDNA template is contaminated by genomic DNAs: Prepare a new cDNA template;

(4) Non-specific amplification tends to occur when the Ct values are ≥ 30;

(5) Increase the annealing temperature (no higher than 63°C);

(6) Increase the template concentration: If the target gene expression is too low, dye-based qPCR is prone to produce primer dimers that affect the assay results. In this case, increase the template concentration.

Q11: The qPCR instrument shows a BadRox error.

A11: The BadRox error may be caused by:

(1) Instrument issues: The instrument needs to be calibrated. The ABI QCR instruments should be calibrated annually, or problems may occur with fluorescent signal acquisition. Engineers should be contacted to calibrate aging instruments or replace parts.

(2) The client uses a small reaction system, such as a 10 μl amplification system, which leads to a low overall ROX concentration and then the BadRox error.

(3) The ROX concentration of the kit is lower than the detection threshold of the instrument.

Q12: Poor experiment repeatability

A12: (1) Inaccurate sample loading volume: Use a more accurate pipette; increase the volume of the reaction system by diluting the template by a higher dilution factor.

(2) Inconsistent temperature control across different locations in the qPCR system: Calibrate the system regularly.

(3) The template concentration is too low. The lower the template concentration, the poorer the repeatability. Decrease the template dilution factor or increase the sample volume.

(4) Design 4-6 replicate wells, discard those with poor repeatability, and use the rest for subsequent analysis.

Q13: Which product is recommended for qPCR of GC-rich templates?

A13: Q111 is recommended for qPCR.

Q14: How should samples be diluted if the reference gene has a low Ct value but the target gene has a high Ct value?

A14: Use a diluted template for qPCR of the reference gene and an undiluted template for qPCR of the target gene. The result can still be calculated using the 2-ΔΔCt method, as the two Ct value subtractions will cancel out the dilution factors. However, make sure that the dilution factor remains the same when different samples are used to assay the same gene.

Q15: How to select the template diluent?

A15: Use the purchased nuclease-free water, DEPC water, or homemade ddH2O to dilute the template. TE contains EDTA, which inhibits enzyme activity, and should not be used as a template diluent.

Q16: How to eliminate aerosol contamination be removed in the system?

A16: (1) Use a new Mix, primers, or templates.

(2) Prepare the reaction system on an ultra-clean workbench if possible to minimize aerosol contamination. Wipe the benchtop of the ultra-clean workbench regularly with diluted 84 disinfectant; clean pipettes with alcohol cotton balls, and irradiate them with UV lamps to eliminate nucleic acid contamination introduced by sample loading over long periods of time.

(3) Do not uncap qPCR products in the sample loading room.

(4) Use Vazyme #R504 RNase, RNA and DNA Remover.

Q17: How to determine the gene expression?

A17: If a gene's Ct value exceeds 30, create the standard curve via gradient dilution of PCR products and determine the primers' amplification efficiency. If the amplification efficiency is 90% – 120%, it means that the high Ct value is caused by low gene expression.

Q18: Poor repeatability among replicate wells.

A18: Poor repeatability among replicate wells generally involves two scenarios:

Scenario 1: When the Ct value is large, such as Ct ≥ 30, poor repeatability is normal. This satisfies the Poisson distribution. That's to say, when there are very few effective templates, the templates randomly bind to primers, leading to large variations in Ct values across replicate wells.

Solution: If the melt curve does not have miscellaneous peaks and the △Ct between the NTC and the target gene is over 5, it means that the Ct values are accurate. Design more replicate wells and choose Ct values of wells with good repeatability for analysis.

Scenario 2: Normal Ct values (such as Ct < 30) but poor repeatability. These are generally related to problematic lab operations.

Solution: Troubleshoot from the following aspects:

① Sample loading accuracy; ② Pipetting accuracy; ③ Regular qPCR system calibration.

Sample loading accuracy

(1) Avoid small loading volumes to reduce sample loading errors. Pre-mix primers, SYBR Green Mix and ddH2O, and increase the template dilution factor to load larger sample volumes. For example, instead of adding 1 μl of cDNA stock solution, dilute the cDNA stock solution 5-fold, add 5 μl of the dilute solution to prepare the reaction system, and bring the system volume to 20 μl with ddH2O.

(2) Mix the SYBR Green Mix and the prepared mix thoroughly. Reagents taken from the fridge at -20°C should be completely thawed and mixed well by inversion. When preparing the reaction system, pipette the Mix up and down to mix it well.

Pipetting accuracy

(1) Calibrate pipettes annually.

(2) Purchase matching tips for pipettes. If tips and pipettes do not match, air leakage is likely to occur when drawing liquids, leading to inaccurate pipetting volumes. When drawing liquids of the same volume, observe whether the liquid levels in the tip are the same.

Regular qPCR system calibration: In general, the qPCR systems are calibrated annually.

Q19: Why is a standard curve constructed for relative quantification?

A19: When the 2-ΔΔCt formula is used to compare gene expression between different samples in relative quantification, the assumption is that the system's amplification efficiency e is close to 100%. The standard curve is constructed in relative quantification to determine whether the system's amplification efficiency e is close enough to 100% to justify using the 2-ΔΔCt formula. If the amplification efficiency e differs substantially from 100%, the actual amplification efficiency should be used to compare gene expression.

Q20: How to perform absolute quantification?

A20: Use a sample with a known copy number/concentration as the standard. Dilute the standard to at least 5 gradient concentrations and simultaneously load the dilute standard and the test sample for qPCR. Construct a standard curve with the Log of the standards' copy numbers as the x-axis and the standards' Ct values as the y-axis. Substitute the Ct value of the test sample into the standard curve equation to derive the copy number/concentration thereof.