Hello all,
I am preparing a diagnostic kit for the detection of viral nucleic acid and testing them using Taqman probes. I am getting initial rise of the curves by around 1000 RFU and then suddenly dropping to zero to give rise to a foxtail-like appearance and thereafter the curves started rising again. This happens in low as well as in high copy number viral nucleic acid samples and in internal control curves as well. How to solve this problem as it is affecting the quantitative values interpretation also. Please help in solving it. I have attached a screenshot of one of the curves obtained.
According to some web sources¹²³⁴⁵, some factors that may cause foxtails are:
- Baseline correction: The foxtails may be an artifact of the automatic baseline correction of the instrument, which may start to calculate the baseline in cycles that show dropping fluorescence. You may want to check the baseline settings and adjust them manually if needed³.
- Dye quenching: The foxtails may be caused by the quenching of the intercalating dye by a proximal G base or by too much internal reference dye (e.g. ROX) in the reaction. You may want to check the dye concentration and the probe design⁴.
- Target heterogeneity: The foxtails may be caused by the amplification of heterogeneous but related DNA targets, which may have different melting temperatures and affect the fluorescence signal. You may want to check the target sequence and the primer specificity⁴.
- Contamination or primer-dimer formation: The foxtails may be caused by unwanted nucleic acid contamination or primer-dimer formation, which may interfere with the amplification of the target sequence. You may want to check the quality and purity of your samples and primers²⁵.
You may want to check these factors in your experiment and see if they are causing the foxtails in your qPCR curves.
(1) RT-PCR / RT-qPCR Troubleshooting - MilliporeSigma. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/troubleshooting.
(2) Troubleshoot your unexpected qPCR amplification curves | IDT. https://www.idtdna.com/pages/education/decoded/article/a-quick-guide-for-troubleshooting-qpcr-data.
(3) Bizzare qRT-PCR amplification curve - Why does signal drop to zero near .... https://www.researchgate.net/post/Bizzare-qRT-PCR-amplification-curve-Why-does-signal-drop-to-zero-near-end-of-run.
(4) Target sequence heterogeneity causes the ‘hook effect ... - BioTechniques. https://www.future-science.com/doi/10.2144/btn-2020-0016.
(5) Why am I getting an abnormal qPCR amplification plot?. https://www.researchgate.net/post/Why-am-I-getting-an-abnormal-qPCR-amplification-plot.
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