A-1   ——Most double-stranded DNA starts melting at 95°C. In some cases, DNA cannot be denatured  well at 90-94°C. Due to partial denaturation, the number of probes and primers participating in the reaction is correspondingly reduced, so the reaction efficiency will decrease. For specific hot-start enzymes, the steps of heat activation should be added correspondingly.

A-2   ——15 to 20 seconds is sufficient for denaturing amplicon. A longer product may take 30 seconds, but a denaturation time of 60 seconds is usually not required.

A-3   ——Design annealing temperature according to Tm value of primer and probe. The annealing time of the three-step SYBR Green is about 20 to 35 seconds and the extension time is 30 to 60 seconds. Double labeled probe usually uses a twostep process, where annealing and extension are combined into one step. The time is about 45 to 60 seconds and the temperature is usually 60°C.

A-4   ——For signal acquisition, SYBR Green should be acquired at 72°C extension, where most of DNA is in double-stranded state. For two-step detection, signal acquisition should be in the integration step of annealing extension. For Taqman method, signals are generally collected at the end of annealing or at the end of extension.

A-1   The instrument does not match the consumables or the detection channel is incorrect.

——Regularly overhaul instruments and replace them with matching consumables.

A-2    The number of reaction cycles is not enough.

——Generally, more than 35 cycles are required, and cycles can be increased according to experimental conditions (for example, 45 cycles), but more than 45 cycles will increase excessive background signals.

A-3    Primers or probes degrade.

——Its integrity can be detected by PAGE electrophoresis. Re-synthesis if it is degraded.

A-4    Improper design of primer or probe.

——The Tm value of the probe should be 5-10°C higher than that of the primer, otherwise the probe will not hybridize but the product has already begun to extend. The difference between Tm values of upstream and downstream primers should not exceed 4°C. Avoid secondary structures in primers or probes.-

A-5    Template degradation.

——Impurities should be avoided in sample preparation and avoid repeated freezing and thawing.

A-1    Low amplification efficiency and the reaction conditions are not optimized enough.

——Refer to Q5.

A-2    Insufficient sample quantity on PCR template or template degradation.

——Increase template quantity to ensure template  integrity.

A-3    Low enzyme activity or the buffer composition changes.

——Pay attention to the validity period and storage conditions of reagents.

A-4    Too long PCR product.

——Generally, a product length of 100-150bp is adopted.

A-1    Errors in sample addition and inaccurate dilution, which make the standard substance not gradient.

——Improve the precision of experimental operation.

A-2    The standard substance degrades.

——Avoid repeated freezing and thawing of the standard, or prepare and dilute the standard again.

A-3   Primers or probes are not good.

——Redesign better primers and probes.

A-4    Inhibitors exist in the template or the template concentration is too high.

——Reduce the template quantity or dilute the template.

A-1    Primers or probes are not designed reasonably.

——Adjust and redesign better primers or probes.

A-2    Some components in the reaction reagent, especially fluorescent dyes, are degraded.

——Check whether the kit is expired or improperly stored. Change to a new kit.

A-3    The reaction conditions are not optimized enough.

——The annealing temperature can be appropriately lowered or changed to a three-step amplification method.

A-4    There are PCR inhibitors in the reaction system.

——It is generally introduced when adding the template. The template should be diluted appropriately and then added into the reaction system to reduce the influence of inhibitors.

A-5   Magnesium ion concentration is low.

——Increase magnesium ion concentration appropriately. Adjust according to the spacing of 0.5 mM.

A-1   The sample addition is not accurate.

——Improve the precision of experimental operation.

A-2   The temperature conditions of the samples on the instrument are different.

——Try to avoid the experiment of sample group in the hole of the outermost circle of the instrument, and correct the instrument regularly.

A-3   The template concentration is low.

——The lower the initial concentration of the sample, the greater the influence of impurities and the worse the repeatability. Educe the dilution multiple of the sample.

A-1   The reagents, enzymes or water used are contaminated.

——Use new reagents and water to clean the experimental area.

A-2   Effect of primer dimer.

——Negative amplification after 35 cycles is normal and can be analyzed with melting curve. If the primer dimer Ct is too forward, the primer should be redesigned.

A-3   Degradation of probe during reaction.

——Use PAGE electrophoresis to detect the probe. Re-synthesis if it is degraded.

A-1 ——The target segment is a short segment, such as miRNA. The Tm value of these products is lower than that of common products.

A-2 ——If the target fragment is a long fragment, it may be the peak of primer dimer, but there is no peak of specific product. Common PCR can be performed with the same template and primer, followed by agarose electrophoresis. Whether specific bands are generated can be judged according to the size of electrophoresis bands, thus adjusting the primer sequence or primer concentration.

A-1  ——The peak is relatively wide and the tip is not sharp enough, which may be due to the influence of the ion ratio in the solution system and also related to the product sequence. Excessive GC content will cause the melting curve to widen. It is generally believed that when the span of the peak does not exceed 7°C, the result is reliable and does not affect the final quantification However, the peak with too large span may be generated by superposition of two amplification products with similar sizes. High concentration (3%-4%) agarose electrophoresis should be performed to detect the presence of non-specific amplification. If so, the melting curve is equivalent to a non-single peak and the result is not credible. Refer to Q “The  melting curve is not a single peak” for adjustment scheme. In addition, it should be confirmed that no deletion mutation will occur in the amplification sequence.

A-2   ——The peak is asymmetric, and the curve slopes of the upper peak and the lower peak are different. The largest possibility is that there is non-specific amplification with different lengths, but the ratio is smaller. Using high concentration agarose electrophoresis, the band may have dispersion. Using three-step amplification and adjusting annealing temperature can effectively improve this situation. If the upper peak curve is relatively gentle, it may be incomplete extension, forming a series of short PCR products. It can be considered to increase the extension time while adjusting the annealing temperature.

A-1    Primer design is not optimized enough.

——Compared with the melting curve of the negative control, it can be judged whether it is primer dimer or non-specific amplification. Adjust primer design accordingly to avoid primer dimer, hairpin structure or nonspecific amplification.

A-2    The primer concentration is not good.

——Appropriately reduce the concentration of primers (200-250 nM is suggested), and pay attention to the concentration ratio of upstream and downstream primers.

A-3    Magnesium ion concentration is too high.

——Appropriately reduce magnesium ion concentration, or select a more appropriate premix kit.

A-4    The template has genomic contamination.

——The introduction of genomic DNA should be avoided during RNA extraction, and the influence of genome amplification should be avoided by designing trans-intron primers.