A rough amplification curve.

The fluorescence signal is too weak and generates after system correction.

Ensure that the dyes premix are not degraded; Replace qPCR specific consumables for a better collection of fluorescent signal.

Amplification curve breaks or slides

The template concentration is relatively high; The endpoint value of the baseline is greater than the Cq value

Reduce baseline endpoint (Cq-4) and reanalyze data.

Sudden drop in amplification curve of individual wells.

There are bubbles in the reaction tube.

Ensure the mix is completely dissolved;

Please do not vortex agitation the mix;

After adding the sample, gently flick the centrifuge tube to remove bubbles;

Extend the pre denaturation time to 10 minutes.

No amplification curve appears after the reaction ends

Too few reaction cycles.

Set the number of cycle to 40, 

but too more will increase too many background signals.

The step of fluorescent signal 

collection is not set or set incorrectly.

The two-step amplification program generally sets signal acquisition at the annealing and extension stages, while the three-step amplification program should set signal acquisition at the 72℃ extension stage.

The primer may have degraded.

Primers that are not used for a long time should be tested for integrity through polyacrylamide gel electrophoresis（PAGE ） to rule out the possibility of degradation.

The template concentration is 

too low.

Reduce the dilution ratio and repeat the experiment. If the sample concentration is unknown, proceed with the experiment from the highest concentration first.

Template is degraded.

Prepare the template again and repeat the experiment.

Cq value appears too late

Low amplification efficiency.

Increase the concentration of primers, try using a three-step amplification, or redesign the primer.

Template concentration is too 

low.

Reduce the dilution ratio and repeat the experiment. If the sample concentration is unknown, proceed with the experiment from the highest concentration first.

Template degraded.

Prepare the template again and repeat the experiment.

Amplification product is too 

long. 

The length of the amplification product should be controlled between 80-200 bp.

Presence of PCR inhibitors.

Generally, it is added to the mix together with the template, increasing the dilution ratio of the template or preparing a high-purity template again.

Signal appears in the blank control.

Pollution of reaction system.

Replace the water used in the blank control. If the same situation persists, continue to replace the primers, pipette tips, PCR tubes, or use a new mix reaction system(prepare in the ultra clean bench to reduce aerosol pollution).

Presence of non-specific amplification such as primer dimer.

It is normal that amplification products appear after 35 cycles in the blank control, and analysis should be carried out in conjunction with the melting curve. Primers should be redesigned or adjust primer concentration or optimize PCR reaction procedures.

Multiple peaks appear on the melting curve.

Poor primer design.

Redesign primers.

The primer concentration is too 

low

Reduce primer concentration appropriately.

Genomic contamination in cDNA templates.

The extracted RNA solution can be treated with DNase, such as dsDNase.

实验重复性差

There may be large errors when adding samples.

Using precise pipettes and high-quality pipette tips for accurate pipetting;

Increase the dilution ratio of the template and reduce sampling errors by adding a large volume of template.

Template concentration is too 

low.

Reduce the dilution ratio and repeat the experiments.

Temperature deviation at different positions of the qPCR instrument.

Regularly calibrate the instruments