Real-Time PCR Data Analysis

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Overview

Real-time PCR has become a widely used technique for a variety of applications, such as gene expression, mutational analysis, and pathogen detection. Many biotechnology companies offer real-time PCR instruments with data analysis software packages to assist with these applications. However, some software packages are easier to use than others, and some offer more features, such as multiple chart/table view and quality control (QC) flags to help enhance the data analysis experience. In this section we give an overview of Bio-Rad's CFX Maestro™ Software features including high resolution melt (HRM) analysis capabilities.

Related Topics: qPCR Instrumentation, qPCR Reagents, qPCR Assay Design and Optimization, Real-Time PCR Experimental Design, MIQE and RDML Guidelines, PCR Troubleshooting, High Resolution Melting and PCR Primer and Probes Chemistries.

 

CFX Maestro Software Features

All of Bio-Rad's real-time PCR detection systems include the powerful, easy-to-use CFX Maestro Software. With one software for all four systems, you can easily open and read files from any of the instruments, collaborate with other researchers, and move to another system without learning to use new software.

Similar to other real-time PCR software, CFX Maestro software offers several analysis modules, including quantification, melt curve, gene expression, allelic discrimination, and end-point analyses. An example of how to analyze a gene expression experiment in CFX Maestro software is shown below.

Set up the plate — indicate position of unknown, no template controls (NTCs), and standard curve samples on plate (Figure 1). This can be done before, during, or after a run.

Fig. 1. Plate setup in the Plate Editor window.

Fig. 1. Plate setup in the Plate Editor window.

Analyze the data — a data file is automatically generated after a run with the gene expression module. Easily view up to six different charts or tables, such as the amplification plot, standard curve, gene expression chart, plate layout, or melt peak with the Custom Data View tab (Figure 2). Check the efficiency and R2 of the standard curve. The efficiency should be within 90–110% and the R2 should be >0.990. If these values are out of range, you will need to troubleshoot your experiment.

Fig. 2. Custom Data View window.

Fig. 2. Custom Data View window.

Export results for publication — Quickly export any charts or tables by right clicking in the window and selecting Save Image As (Figure 3) or Export to Excel (Figure 4). You can also create reports or real-time PCR data markup language (RDML) files for quick import into qbase+ software.

Fig. 4. Select Save Image As in the data analysis window. Fig. 3. Select Export to Excel in the data analysis window.

Fig. 3. Select Save Image As in the data analysis window.


Fig. 4. Select Export to Excel in the data analysis window.

 

High Resolution Melt (HRM) Analysis

In addition to the typical software packages, some instruments also offer HRM software to genotype samples based on their DNA thermal denaturation properties. HRM analysis requires careful experimental design, HRM-specific reagents, and a sensitive real-time PCR instrument with software that can handle large quantities of data. HRM software is usually straightforward, and should allow users to:

  • Compare and combine data from multiple experiments by combining run results into a single melt study
  • Display a plate view for easy identification of sample genotypes
  • Share analysis settings among experiments
  • Analyze multiple experiments from a single plate
  • View all charts in a single window for simplified data analysis and interpretation

An example of analyzing HRM data using Bio-Rad's HRM software, Precision Melt Analysis™ software, is shown below. In this example, Precision Melt Analysis software is used to discriminate between SNP genotypes (C to T substitution). The software uses the default analysis settings to assign a cluster to each sample. A normalized melt curve and a difference curve are generated for each well. These profiles can then be used to determine the different genotypes.

Melt Curve.

Normalized Melt curve.

Difference Curve.

Difference curve.

 

Related Content

 
Literature
Number Description Download
6009 High Resolution Melt Parameter Considerations for Optimal Data Resolution, Rev A Click to download
5859 A Practical Approach to RT-qPCR — Publishing Data That Conform to the MIQE Guidelines, Ver E Click to download
10010421 CFX Manager™ Software Plate for the CFX96™ System, Quick Guide, Rev C Click to download
10010422 CFX Manager™ Software Data Analysis for the CFX96™ System, Quick Guide, Rev C Click to download
10010423 CFX Manager™ Software Gene Expression Analysis for the CFX96™ System, Quick Guide, Rev D Click to download
 
 
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Many biotechnology companies offer real-time PCR instruments with data analysis software packages to assist with these applications. However, some software packages are easier to use than others, and some offer more features, such as multiple chart/table view and quality control (QC) flags to help enhance the data analysis experience. In this section we give an overview of Bio-Rad's <a href="/en-us/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments">CFX Maestro&trade; Software</a> features including high resolution melt (HRM) analysis capabilities.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSO5YMNI">qPCR Instrumentation</a>, <a href="/evportal/destination/solutions?catID=LUSO6XKSY">qPCR Reagents</a>, <a href="/evportal/destination/solutions?catID=LUSO7RIVK">qPCR Assay Design and Optimization</a>, <a href="/evportal/destination/solutions?catID=LUSNJVIVK">Real-Time PCR Experimental Design</a>, <a href="/evportal/destination/solutions?catID=LUSO8N4EH">MIQE and RDML Guidelines</a>, <a href="/evportal/destination/solutions?catID=LUSO3HC4S">PCR Troubleshooting</a>, <a href="/evportal/destination/solutions?catID=LUSOIH97Q">High Resolution Melting</a> and <a href="/evportal/destination/solutions?catID=LUSOJW3Q3">PCR Primer and Probes Chemistries</a>.</p> CFX Maestro Software Features <p>All of Bio-Rad's <a href="/evportal/destination/solutions?catID=LUSO5YMNI">real-time PCR detection systems</a> include the powerful, easy-to-use <a href="/en-us/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments">CFX Maestro Software</a>. 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Plate setup in the Plate Editor window.</strong></p> <p><strong> Analyze the data</strong> &mdash; a data file is automatically generated after a run with the gene expression module. Easily view up to six different charts or tables, such as the amplification plot, standard curve, gene expression chart, plate layout, or melt peak with the Custom Data View tab (Figure 2). Check the efficiency and R<sup>2</sup> of the <a href="/evportal/destination/solutions?catID=LUSO7RIVK">standard curve</a>. The efficiency should be within 90&ndash;110% and the R<sup>2</sup> should be &gt;0.990. If these values are out of range, you will need to <a href="/evportal/destination/solutions?catID=LUSOBDHYP">troubleshoot</a> your experiment.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt25_img2.jpg" alt="Fig. 2. Custom Data View window." width="580" height="350" /></p> <p class="caption"><strong>Fig. 2. Custom Data View window.</strong></p> <p><strong> Export results for publication</strong> &mdash; Quickly export any charts or tables by right clicking in the window and selecting Save Image As (Figure 3) or Export to Excel (Figure 4). You can also create reports or real-time PCR data markup language (RDML) files for quick import into qbase+ software.</p> <table border="0" width="100%"> <tbody> <tr> <td><span style="width: 290px; float: left;"><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt25_img4.gif" alt="Fig. 4. Select Save Image As in the data analysis window." width="288" height="304" /></span></td> <td><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt25_img3.gif" alt="Fig. 3. Select Export to Excel in the data analysis window." width="269" height="304" /></td> </tr> <tr> <td><br /> <p class="caption"><strong>Fig. 3. Select Save Image As in the data analysis window.</strong></p> </td> <td><br /> <p class="caption"><strong>Fig. 4. Select Export to Excel in the data analysis window.</strong></p> </td> </tr> </tbody> </table> <div class="top"><a href="#helptop">Back to Top</a></div> High Resolution Melt (HRM) Analysis <p>In addition to the typical software packages, some instruments also offer <a href="/evportal/destination/solutions?catID=LUSOIH97Q">HRM</a> software to genotype samples based on their DNA thermal denaturation properties. HRM analysis requires careful experimental design, HRM-specific reagents, and a sensitive real-time PCR instrument with software that can handle large quantities of data. HRM software is usually straightforward, and should allow users to:</p> <ul> <li>Compare and combine data from multiple experiments by combining run results into a single melt study</li> <li>Display a plate view for easy identification of sample genotypes</li> <li>Share analysis settings among experiments</li> <li>Analyze multiple experiments from a single plate</li> <li>View all charts in a single window for simplified data analysis and interpretation</li> </ul> <p>An example of analyzing HRM data using Bio-Rad's HRM software, <a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=df190aee-f184-497e-bfb6-b6dd632c99b5">Precision Melt Analysis&trade; software</a>, is shown below. In this example, Precision Melt Analysis software is used to discriminate between SNP genotypes (C to T substitution). The software uses the default analysis settings to assign a cluster to each sample. A normalized melt curve and a difference curve are generated for each well. These profiles can then be used to determine the different genotypes.</p> <p><img style="margin-bottom:-20px; margin-left: -10px" src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt25_img5.jpg" alt="Melt Curve." width="401" height="227" /></p> <p class="caption"><strong>Normalized Melt curve.</strong></p> <p><img style="margin-bottom:-15px; margin-left: -10px" src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/gxt25_img6.jpg" alt="Difference Curve." width="357" height="213" /></p> <p class="caption"><strong>Difference curve.</strong></p> <div class="top"><a href="#helptop">Back to Top</a></div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="CFX Manager Software Tutorial" onclick="window.open('/webroot/web/movies/lsr/products/amplification_pcr/product_overlay_content/global/cfx_manager_tutorial/cfx_manager_tutorial.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=1060, height=820');" href="javascript:void(0);"><img style="border:none;" src="http://www.biorad-ads.com/solutions/webroot/web/images/lsr/support/tutorials/global/cfx_tutorial_th.jpg" alt="" /></a></div> <div class="videoDesc"><br /><a title="CFX Manager Software Tutorial" onclick="window.open('/webroot/web/movies/lsr/products/amplification_pcr/product_overlay_content/global/cfx_manager_tutorial/cfx_manager_tutorial.html', 'StatusBar', 'toolbar=no, resizable=no, scrollbars=no, width=1060, height=820');" href="javascript:void(0);">CFX Manager Software Tutorial</a><br /></div> <div class="clear">&nbsp;</div> </div> 6009 5859 10010421 10010422 10010423 Related Websites <ul class="rightarrow"> <li><a class="subaccord" href="https://www.qbaseplus.com/" target="_blank">qbase+ Software</a></li> <li><a class="subaccord" href="http://data-analysis.gene-quantification.info/" target="_blank">Data Analysis Introduction</a></li> </ul> Life Science Research/Products/Amplification - PCR/Amplification Software/CFX Manager Software (Version 1.1) ->MT::aed9803d-cb4d-4ecb-9263-3efc9e650edb##Life Science Research/Products/Amplification - PCR/Amplification Software/Precision Melt Analysis Software ->MT::df190aee-f184-497e-bfb6-b6dd632c99b5##Life Science Research/Products/Amplification - PCR/Real-Time PCR Detection Systems/CFX Connect Real Time PCR Detection System ->MTS::LN5TFG15##Life Science Research/Products/Amplification - PCR/Real-Time PCR Detection Systems/CFX96 Touch Real-Time PCR Detection System ->MTS::LJB1YU15##Life Science Research/Products/Amplification - PCR/Real-Time PCR Detection Systems/CFX384 Touch Real-Time PCR Detection System ->MTS::LJB22YE8Z## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/MIQE and RDML ->MTS::LUSO8N4EH##Life Science Research/Solutions/Technologies/PCR/Assay design and optimization ->MTS::LUSO1VKG4##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Reagents ->MTS::LUSO6XKSY## Eddie C Real-Time PCR Data Analysis Discover the features and capabilities of CFX Manager Software for qPCR data analysis in gene expression, mutational analysis, and pathogen detection assays. pcr, qpcr, real-time pcr, realtime, rt-pcr, qpcr analysis, qpcr assays, data analysis, pcr software, real-time analysis, quantitative pcr 12/28/11 10:03 AM 12/28/21 10:04 AM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/qPCR_Real_Time_PCR N 0
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