Troubleshooting

 

grafica kitpcr1

grafica kitpcr2

 

 

 

 

 

 

 

Observations and recommendations

  • If amplification plot shows a low magnitude curve, you can modify the scale in plot property settings and better observe the curve shape.
  • If the curves show a sigmoidal shape, it is considered as positive.
  • Low efficiency may be due pipetting errors, presence of qPCR inhibitors or reagent expiration. If possible repeat the assay.

grafica kitpcr3

grafica kitpcr4

 

Observations and recommendations

  • Depending of quality of stored samples, quality of RNA extraction, viral load or stage of infection, some genes may not amplify.
  • If possible, repeat RNA extraction of the sample. For the results interpretation must consider the negative control profile.

grafica kitpcr5

Observations and recommendations

  • Generally the curves of viral target exceed the curve of the internal control.
  • If the viral target curve is lower than internal control curve, but show sigmoidal shape, it is considered a valid amplification.

  • In multiplex qPCR systems it could happen due to competition among probes or/and other components of the assay.

grafica kitpcr7

 

Observations and recommendations

  • In the same experiment run differences in the curvemagnitude could be obtained.
  • This corresponds to differences in the quality of extraction of RNA or viral load.
  • If the curves show sigmoidal shape, it is considered as positive.

grafica kitpcr8

grafica kitpcr9

Observations and recommendations

  • If some target amplifies in the negative control well, check the curve shape.

  • If present, sigmoidal shape could be due to contamination of the negative control or pipetting errors. Repeat the assay and change the negative control.

  • If not present, sigmoidal shape could be due to internal assay conditions or related to reactives storage. Considering this as background noise, you can modify the threshold line in the analysis settings of the software, it may help to interpret and discriminate the curve profiles.

grafica kitpcr10

Observations and recommendations

  • If the positive control does not amplify, it may be due to problems with the mix preparation, incorrect pipetting, integrity of the control and qPCR program setting.

  • If possible, repeat the assay and make sure that assay conditions are correct. You may change the positive control and check the integrity of qPCR reagents.

 

grafica kitpcr11

 

Observations and recommendations

  • The inconsistency of replicate results may be due pipetting errors or incorrect calibration of qPCR instrument.

  • Consider calibrating the pipettes and qPCR instrument and repeating the assay.
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