{"id":1162,"date":"2020-06-17T00:36:17","date_gmt":"2020-06-17T00:36:17","guid":{"rendered":"https:\/\/www.idtdna.com\/page\/a-quick-guide-for-troubleshooting-qpcr-data"},"modified":"2026-07-29T21:18:12","modified_gmt":"2026-07-29T21:18:12","slug":"a-quick-guide-for-troubleshooting-qpcr-data","status":"publish","type":"post","link":"https:\/\/www.idtdna.com\/page\/support-and-education\/decoded-plus\/a-quick-guide-for-troubleshooting-qpcr-data\/","title":{"rendered":"Troubleshooting Your qPCR Data and Amplification Curves"},"content":{"rendered":"<p>Good qPCR amplification curves should look like Figure 1. During probe-based qPCR assays, fluorescence is released from the probe as DNA polymerase copies the template or target nucleic acid sequence. The exonuclease activity of thermostable DNA polymerase hydrolyzes the probe, thus separating its 5' fluorophore from the quencher. The fluorescence of the degraded probe is divided by the fluorescence of an internal reference dye, ROX, to find the normalized fluorescence value, Rn. This value is then plotted for each cycle (Figure 1A).<\/p>\n<p>Once the fluorescence values increase enough to reach the detection threshold of the instrument, the fluorescence will continue to accumulate for each cycle until the amount of the amplicon competes with the primers for hybridization, and a plateau in fluorescence accumulation is reached.<\/p>\n<p>In Figure 1B, the intersection of the amplification curve and the threshold is defined as the C<sub>q<\/sub>\u00a0value, an approximate measure of the concentration of target sequences in the sample. The C<sub>q<\/sub>\u00a0values are evaluated relative to an internal control gene or to a standard curve created with serial dilutions of a known amount of target sample.<\/p>\n<figure class=\"wp-block-image\">\n    <img decoding=\"async\" title=\"Art291-PCR-Guide Pt 3 Troubleshooting Fig 1\" src=\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/741e7f15-3279-6e2e-aa53-ff00001c1b3c-artboard-18.png\" alt=\"Ideal or expected qPCR amplification curve data\" data-displaymode=\"Original\" \/><figcaption class=\"image-caption\">Figure 1. Ideal qPCR amplification results. (A) Linear scale view for fluorescence (Rn) x cycle number. (B) Log scale view of \u0394Rn (shown on a log<sub>10<\/sub> scale) x cycle number.<\/figcaption><\/figure>\n<p>qPCR is a complex, multifaceted process, and several factors can cause suboptimal amplification. So sometimes your curves look like these (Figures 2 and 3):<\/p>\n<figure class=\"wp-block-image\"><img decoding=\"async\" title=\"Art291-PCR-Guide Pt 3 Troubleshooting Fig 2\" src=\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/741e7f15-3279-6e2e-aa53-ff00001c1b3c-fig-2.png\" alt=\"Suboptimal qPCR amplification curves\" data-displaymode=\"Original\" \/><figcaption class=\"image-caption\">Figure 2. Suboptimal qPCR amplification curves. (A) Fluorescent signals increase too early and may be the result of too much template. (B) When the baseline is incorrectly defined from the early-cycle fluorescence values, the \u0394Rn signal barely exceeds the threshold.<\/figcaption><\/figure>\n<figure class=\"wp-block-image\"><img decoding=\"async\" title=\"Art291-PCR-Guide Pt 3 Troubleshooting Fig 3\" src=\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/741e7f15-3279-6e2e-aa53-ff00001c1b3c-fig-3.png\" alt=\"Suboptimal qPCR amplification curves\" data-displaymode=\"Original\" \/><figcaption class=\"image-caption\">Figure 3. Suboptimal qPCR amplification curves. (A) and (B) No fluorescent signal is recorded.<\/figcaption><\/figure>\n<p>You might observe something very different from what you expect (Figures 4-7):<\/p>\n<figure class=\"wp-block-image\"><img decoding=\"async\" title=\"Art291-PCR-Guide Pt 3 Troubleshooting Fig 4\" src=\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/741e7f15-3279-6e2e-aa53-ff00001c1b3c-fig-6.png\" alt=\"qPCR amplification curves of low height\" data-displaymode=\"Original\" \/><figcaption class=\"image-caption\">Figure 4. Lower than expected height of amplification curve. This can be caused by dye quenching by a proximal G base, differences in probe concentration or the master mix used, too much internal reference dye (e.g. ROX) in the reaction, or incorrect instrument calibration.<\/figcaption><\/figure>\n<figure class=\"wp-block-image\"><img decoding=\"async\" title=\"Art291-PCR-Guide Pt 3 Troubleshooting Fig 5\" src=\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/741e7f15-3279-6e2e-aa53-ff00001c1b3c-fig-7.png\" alt=\"Amplification of the No-Template Control (NTC)\" data-displaymode=\"Original\" \/><figcaption class=\"image-caption\">Figure 5. Amplification of the no-template control (NTC). Amplification of the NTC is often a sign of unwanted nucleic acid contamination or primer-dimer formation.<\/figcaption><\/figure>\n<figure class=\"wp-block-image\"><img decoding=\"async\" title=\"Art291-PCR-Guide Pt 3 Troubleshooting Fig 6\" src=\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/741e7f15-3279-6e2e-aa53-ff00001c1b3c-fig-10.png\" alt=\"qPCR dilution series produce inconsistent Cq intervals\" data-displaymode=\"Original\" \/><figcaption class=\"image-caption\">Figure 6. qPCR sample dilution series produces inconsistent Cq intervals. Unexpected Cq intervals can be caused by contaminants in the sample. These can derive from the host tissue or cells, or the enzymes used in the reverse transcriptase reaction or qPCR. In addition, technical issues such as improper dilution calculation or pipetting errors can produce inconsistent data.<\/figcaption><\/figure>\n<figure class=\"wp-block-image\"><img decoding=\"async\" title=\"Art291-PCR-Guide Pt 3 Troubleshooting Fig 7\" src=\"https:\/\/www.idtdna.com\/page\/wp-content\/uploads\/idt-images\/741e7f15-3279-6e2e-aa53-ff00001c1b3c-fig-11.png\" alt=\"Amplification curves with delayed Cq values\" data-displaymode=\"Original\" \/><figcaption class=\"image-caption\">Figure 7. Replicate amplification curves with delayed Cq values. Partial degradation of the nucleic acid templates or pipetting errors can result in inconsistent replicates.<\/figcaption><\/figure>\n<p>Thus, troubleshooting or generation of additional data may be required to achieve optimal qPCR results. But first you need to determine what is causing the flawed data.<\/p>\n<p>The\u00a0<em><a href=\"https:\/\/go.idtdna.com\/qPCR-guide-part-3.html\" target=\"_blank\" rel=\"noopener\">Real-time qPCR guide: Part 3\u2014troubleshooting<\/a>\u00a0<\/em>uses stylized drawings like the above and real amplification plots to help you identify the factors that could be compromising your results. These might include: sample degradation, low target copy number, incorrectly assigned dye detector, or overlapping fluorescent emission spectra. Just match your data to one of the images that best represents your own data and refer to the indicated guide section to learn what can cause such amplification curves and how to improve them. Click here to register to download the\u00a0<em><a href=\"https:\/\/go.idtdna.com\/qPCR-guide-part-3.html\" target=\"_blank\" rel=\"noopener\">Real-time qPCR guide: Part 3\u2014troubleshooting<\/a>.\u00a0<\/em><\/p>\n<p><strong>For research use only.\u00a0Not for use in diagnostic procedures.<\/strong>\u00a0Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Doc ID: RUO23-1689_001<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Good qPCR amplification curves should look like Figure 1. During probe-based qPCR assays, fluorescence is released from the probe as DNA polymerase copies the template or target nucleic acid sequence. The exonuclease activity of thermostable DNA polymerase hydrolyzes the probe, thus separating its 5' fluorophore from the quencher. The fluorescence of the degraded probe is [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"ct_builder_shortcodes":"","ct_template_type":"","ct_parent_template":0,"inline_featured_image":false,"footnotes":""},"class_list":["post-1162","post","type-post","status-publish","format-standard","hentry"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.0 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Troubleshooting qPCR Amplification Curves | IDT<\/title>\n<meta name=\"description\" content=\"Get expert tips for troubleshooting qPCR amplification curves and resolving common issues to improve accuracy and consistency.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" 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