Real-Time PCR Troubleshooting

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Overview

Are you having trouble with your real-time PCR experiment? Do you need help diagnosing your experiment? The Real-Time PCR Doctor is here to help. Unexpected fluorescence data are symptomatic of problems with your real-time PCR reaction components or amplification protocol. Click one of the symptoms below to learn about possible causes and treatments.

Related Topics: What Is Real-Time PCR?, How Real-Time PCR Works, and qPCR Assay Design and Optimization.

 

 

 

 

Allelic Discrimination Experiments

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High Resolution Melt (HRM) Experiments:

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If you don't see your symptom listed above, please contact technical support at 1-800-424-6723, option 2.


Further Reading

Hu CJ et al. (2006). Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol 26, 3514–3526. PMID: 16611993

Ivanova N et al. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538. PMID: 16767105

Li Y et al. (2006). DAPK1 variants are associated with Alzheimer's disease and allele-specific expression. Hum Mol Genet 15, 2560–2568. PMID: 16847012

Morozumi M et al. (2006). Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes. J Clin Microbiol. 44, 1440–1446. PMID: 16597874

Ridd K et al. (2006). Association of gene expression with sequential proliferation, differentiation, and tumor formation in murine skin. Carcinogenesis 27, 1556–1566. PMID: 16537558

 

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Quantitative PCR (qPCR): Validating a SYBR® Green I Assay
This tutorial describes the characteristics of an optimized SYBR Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.
 
Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay
This tutorial describes the characteristics of an optimized SYBR® Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.
 
Data Analysis: Real-Time Quantitative PCR
This tutorial pertains to the analysis of real-time quantitative PCR (qPCR) data.
 
Literature
Number Description Download
7216 End the Cycle of Repeated, Failed Experiments with a Robust Field Guide to qPCR Click to download
6894 Tips, Tricks & Best Practices: The Ultimate qPCR Assay Design Guide Click to download
5692 Real-Time qPCR as a Tool for Evaluating RNAi-Mediated Gene Silencing, Rev A Click to download
6004 A Practical Guide to High Resolution Melt Analysis Genotyping, Rev A Click to download
6009 High Resolution Melt Parameter Considerations for Optimal Data Resolution, Rev A Click to download
6047 Optical Design of CFX96™ Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye, Rev A Click to download
5962 Fast Optimization of a Multiplex Influenza Identification Panel Using a Thermal Gradient, Rev A Click to download
 
 
LUSOBDHYP [referer] = [https://www.bio-rad.com/en-us/applications-technologies/real-time-pcr-troubleshooting] [x-forwarded-proto] = [https] [x-forwarded-port] = [443] [x-forwarded-for] = [104.197.19.126, 10.232.18.66] [accept] = [text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8] [seourl] = [/en-us/applications-technologies/real-time-pcr-troubleshooting] [x-amzn-trace-id] = [Root=1-5d83b65a-25c8b9b0f567b7a0d2e94854] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=LUSOBDHYP] [host] = [10.232.17.28:1776] [x-request-uri] = [/en-us/applications-technologies/real-time-pcr-troubleshooting] [connection] = [Keep-Alive] [user-agent] = [Mozilla/5.0 (compatible; heritrix/3.3.0-SNAPSHOT-20150302-2206 +http://127.0.0.1)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/real-time-pcr-troubleshooting LSR LUSOBDHYP Troubleshooting Real-Time PCR Troubleshooting /webroot/web/html/lsr/solutions/technologies/qpcr_real-time_pcr <script type="text/javascript">// <![CDATA[ $('head').append('<meta name="DCSext.at_banner" content="Real-Time PCR Troubleshooting" /><meta name="DCSext.at_banner_e" content="v" />'); $(document).ready(function () { $("#rightcol").append('<div id="relatedProducts">&nbsp;</div>'); $('#relatedProducts').load('/webroot/web/html/lsr/solutions/technologies/pcr/related-products-a-t-rtpcr-troubleshooting.html'); }); // ]]></script> <p>Are you having trouble with your real-time PCR experiment? Do you need help diagnosing your experiment? The <strong>Real-Time PCR Doctor</strong> is here to help. Unexpected fluorescence data are symptomatic of problems with your real-time PCR reaction components or amplification protocol. Click one of the symptoms below to learn about possible causes and treatments.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSO4W8UU#1">What Is Real-Time PCR?</a>, <a href="/evportal/destination/solutions?catID=LUSO4W8UU#2">How Real-Time PCR Works</a>, and <a href="/evportal/destination/solutions?catID=LUSO7RIVK">qPCR Assay Design and Optimization</a>.</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p></p> <div id="didYouknow"> <script type="text/javascript">// <![CDATA[ $('#didYouknow').load('/webroot/web/html/lsr/products/amplification_pcr/did-you-know-primepcr.html'); // ]]></script> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Gene Expression/Quantification Experiments:</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel1"> <img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img1.jpg" border="0" alt="Poor Signal or No Signal" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel1">Poor signal or no signal</a><br />&nbsp;</td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img2.jpg" border="0" alt="Signal in Negative Control" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel2">Signal in negative control</a><br />&nbsp;</td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img3.jpg" border="0" alt="CT Values Differing Across Replicate Samples" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel3">Poor reproducibility across replicate samples</a></td> <td width="25%"><a href="/evportal/destination/solutions?catID=LUSOCICZF#gel4"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img4.jpg" border="0" alt="Low or High Reaction Efficiency" width="110" height="110" /></a><br /> <a href="/evportal/destination/solutions?catID=LUSOCICZF#gel4">Low or high reaction efficiency</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Allelic Discrimination Experiments</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="50%"><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img5.jpg" border="0" alt="More than 3 clusters" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel1">More than 3 clusters</a></td> <td width="50%"><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img6.jpg" border="0" alt="Homozygotes as heterozygotes" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOFGDN#gel2">Homozygotes as heterozygotes</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"> <div class="contentroundtop"> <h3><strong>High Resolution Melt (HRM) Experiments</strong>:</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <p><strong>My experiment displays:</strong></p> <table class="pd_table pd_centeredcell" border="0"> <tbody> <tr> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel1"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img7.jpg" border="0" alt="Missed call or clustering" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel1">Missed call or clustering</a></td> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel2"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img8.jpg" border="0" alt="Amplification did not reach plateau" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel2">Amplification did not reach plateau</a></td> <td width="32%"><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel3"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt26_img9.jpg" border="0" alt="Poor amplification or failed reaction" width="110" height="110" /></a><br /><a href="/evportal/destination/solutions?catID=LUSOGO2B7#gel3">Poor amplification or failed reaction</a></td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <p style="margin-top: 10px;">If you don't see your symptom listed above, please contact technical support at 1-800-424-6723, option 2.</p> <br /> <a name="further_reading"></a> <h3>Further Reading</h3> <img style="height: 1px; margin: 5px 0px 15px; width: 620px;" src="http://www.bio-rad.com/webroot/web/images/1x1grey.gif" alt="" /> <p>Hu CJ et al. (2006). Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol 26, 3514&ndash;3526. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16611993" target="_blank" rel="noopener noreferrer">16611993</a></p> <p>Ivanova N et al. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533&ndash;538. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16767105" target="_blank" rel="noopener noreferrer">16767105</a></p> <p>Li Y et al. (2006). DAPK1 variants are associated with Alzheimer's disease and allele-specific expression. Hum Mol Genet 15, 2560&ndash;2568. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16847012" target="_blank" rel="noopener noreferrer">16847012</a></p> <p>Morozumi M et al. (2006). Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes. J Clin Microbiol. 44, 1440&ndash;1446. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16597874" target="_blank" rel="noopener noreferrer">16597874</a></p> <p>Ridd K et al. (2006). Association of gene expression with sequential proliferation, differentiation, and tumor formation in murine skin. Carcinogenesis 27, 1556&ndash;1566. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16537558" target="_blank" rel="noopener noreferrer">16537558</a></p> <div class="top"><a href="#helptop">Back to Top</a></div> <p> <script src="http://admin.brightcove.com/js/BrightcoveExperiences.js" type="text/javascript"></script> </p> <div class="videowrap"> <div class="videoImg"><a title="PCR Fundamentals" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pcr_fundamentals.html' );" href="javascript:void(0);"><img style="border: none;" src="/webroot/web/images/lsr/support/tutorials/global/ov_pcr_fundamentals.jpg" alt="PCR Fundamentals" /></a></div> <div class="videoDesc"><br /><a title="PCR Fundamentals" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_pcr_fundamentals.html' );" href="javascript:void(0);">PCR Fundamentals</a><br /> &nbsp;<br />&nbsp;</div> <div class="clear">&nbsp;</div> </div> <p> <script type="text/javascript">// <![CDATA[ brightcove.createExperiences(); // ]]></script> </p> <div class="videowrap"> <div class="videoImg"><a title="Performing Fast PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_fast_pcr.html' );" href="javascript:void(0);"><img style="border: none;" src="/webroot/web/images/lsr/support/tutorials/global/ov_fast_pcr.jpg" alt="Performing Fast PCR" /></a></div> <div class="videoDesc"><br /><a title="Performing Fast PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/support/ov_fast_pcr.html' );" href="javascript:void(0);">Performing Fast PCR</a><br /> &nbsp;<br />&nbsp;</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Quantitative PCR (qPCR): Validating a SYBR Green I Assay" onclick="window.open('/webroot/web/html/lsr/support/qPCR_Assay_Validation.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border: none;" src="/webroot/web/images/lsr/support/tutorials/global/qpcr_validating_qybr_green.png" alt="" /></a></div> <div class="videoDesc"><a title="Quantitative PCR (qPCR): Validating a SYBR Green I Assay" onclick="window.open('/webroot/web/html/lsr/support/qPCR_Assay_Validation.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Quantitative PCR (qPCR): Validating a SYBR<sup>&reg;</sup> Green I Assay</a><br /> This tutorial describes the characteristics of an optimized SYBR Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay" onclick="window.open('/webroot/web/movies/lsr/support/qPCR_Hallmarks.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border: none;" src="/webroot/web/images/lsr/support/tutorials/global/qpcr_hallmark.png" alt="" /></a></div> <div class="videoDesc"><a title="Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay" onclick="window.open('/webroot/web/movies/lsr/support/qPCR_Hallmarks.htm', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Quantitative PCR (qPCR): Hallmarks of an Optimized SYBR Green I Assay</a><br /> This tutorial describes the characteristics of an optimized SYBR<sup>&reg;</sup> Green I quantitative PCR (qPCR) assay. qPCR assays must be optimized to ensure results that are biologically and statistically significant. Topics include a brief review of qPCR chemistry, with an emphasis on SYBR Green I reactions, and definitions of the four main characteristics, or hallmarks, of an optimized qPCR assay.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="Data Analysis: Real-Time Quantitative PCR" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/data_analysis/data_analysis.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);"><img style="border: none;" src="http://www.biorad-ads.com/solutions/webroot/web/images/lsr/support/tutorials/global/ov_data_analysis_qpcr.gif" alt="" /></a></div> <div class="videoDesc"><a title="Data Analysis: Real-Time Quantitative PCR" onclick="window.open('http://bio-rad.centra.com/bio-radroot/learning%20objects/data_analysis/data_analysis.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=810, height=575');" href="javascript:void(0);">Data Analysis: Real-Time Quantitative PCR</a><br />This tutorial pertains to the analysis of real-time quantitative PCR (qPCR) data.</div> <div class="clear">&nbsp;</div> </div> 7216 templatedata/internet/documentation/data/LSR/Literature/7216_1563396055.xml 6894 templatedata/internet/documentation/data/LSR/Literature/6894_1559950197.xml 5692 6004 6009 6047 5962 Related Websites <ul class="rightarrow"> <li><a class="subaccord" href="http://evolution.gene-quantification.info/" target="_blank" rel="noopener noreferrer">Real-Time PCR</a></li> </ul> Life Science Research/Products/Nucleic Acid Sample Preparation and Purification/RNA Isolation/Aurum Total RNA 96 Kit ->MT::49da5896-dfde-4292-8021-9df1d8b77fec##Life Science Research/Products/Nucleic Acid Sample Preparation and Purification/DNA Isolation/Quantum Prep Plasmid Kits ->MT::7167c5db-d618-47a5-9185-9c612fe4fbe0##Life Science Research/Products/Amplification - PCR/PCR Reagents/Reverse Transcription Reagents/iScript Advanced cDNA Synthesis Kit ->MTS::LIXR5J15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Supermixes for PCR and Real-Time PCR/SsoAdvanced SYBR Green Supermix ->MTS::LML5JY15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Supermixes for PCR and Real-Time PCR/Precision Melt Supermix ->MTS::LNIQ3X15## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Assay Design and Optimization ->MTS::LUSO7RIVK##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Reagents ->MTS::LUSO6XKSY##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Data analysis ->MTS::LUSOA9LPT##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/High Resolution Melting -HRM- ->MTS::LUSOIH97Q##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/MIQE and RDML ->MTS::LUSO8N4EH## Eddie C Real-Time PCR Troubleshooting Having trouble with your real-time PCR experiments? 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