Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye

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en-us MW472W15 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye /webroot/web/html/lsr/solutions/technologies/qpcr_real-time_pcr <p>Certain real-time PCR instruments require an internal reference dye for fluorescent signal normalization and correction of well-to-well optical variations. Variation in fluorescence can result from instrument design. ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization throughout the real-time qPCR assay.</p> <p>Related Topics: <a href="/en-us/applications-technologies/qpcr-instrumentation">qPCR Instrumentation</a>, <a href="/en-us/applications-technologies/qpcr-real-time-pcr-reagents">qPCR/Real-Time PCR Reagents</a>, and <a href="/en-us/applications-technologies/qpcr-assay-design-optimization">qPCR Assay Design and Optimization</a>.</p> <br /> <!-- <div style="border: 1px solid #cccccc; width: 614px; height: 49px; margin-top: 20px; margin-bottom: 20px;"> <div style="float: left; width: 123px; height: 49px; background-color: #333333; margin-right: 10px; padding-left: 14px;"> <div style="font-size:13px; color:#FFF; margin-top: .8em;" mce_style="font-size:13px; color:#FFF; margin-top: .8em;"><b>WHY IS MY SUPERMIX PINK?</b></div> </div> <div style="margin-top: .9em; padding-right:5px" mce_style="margin-top: .9em; padding-right:5px"><b>Bio-Rad&rsquo;s universal real-time PCR supermixes include a new pink universal passive reference dye that is suitable for use on any qPCR system. <a style="font-weight:bold" mce_style="font-weight:bold" href="/en-us/faq/Why-is-my-supermix-p_1384537055/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye" mce_href="/en-us/faq/Why-is-my-supermix-p_1384537055/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">Find out why</a></b></div> </div> --> Understanding Cq Values, Optical Systems, and Passive Reference Dyes <p>A Cq (quantification cycle) value is an exponential number derived from the intersection of the threshold and a given amplification curve and is a relative concentration measurement of the target presence in the qPCR reaction. Several factors independent of the sample influence the Cq value &mdash; the optical system, PCR supermix, threshold and baseline settings, and the presence or absence of a passive reference dye such as ROX. Before explaining how a passive reference dye like ROX impacts Cq values and qPCR data, it is important to understand how optical real-time PCR detection systems work.</p> <p>The optical design of most real-time PCR systems, using a lamp or other single stationary light source, creates illumination and detection light paths that vary for each well of the thermal cycler block. The variation in light path lengths produces different absolute fluorescence measurements for wells that contain the same concentration of a reporter fluorophore. A well with a shorter light path, typically located in the middle of the block, will have a higher fluorescence reading compared to a well with a longer light path, typically located on the perimeter of the block (Figure 1).</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/light-path-differences-between-wells-of-a-lamp-based-real-time-pcr-system.jpg" alt="Light path differences between wells of a lamp-based real-time PCR system" width="423" height="255" /></p> <p class="caption"><strong>Fig. 1. Light path differences between wells of a lamp-based real-time PCR system.</strong> The well in the center of the block has a shorter light path and, subsequently, emits a higher fluorescence signal compared to the well on the perimeter, which has a longer light path.</p> <p>The differences in absolute fluorescence levels do not impact real-time PCR results because results are determined by measuring the change in fluorescence of a reporter measured over the course of the reaction. For real-time PCR data processing, the sample fluorescence for each well is first baseline subtracted and then the quantification cycle (Cq) value is determined as a cycle number at which fluorescence has increased above background.</p> <p>&nbsp;</p> Normalization of Fluorescence Data Using ROX <p>Some lamp- and laser-based real-time PCR systems utilize an internal reference dye to normalize well-to-well fluorescence signal differences resulting from optical path length variations. Normalizing the fluorescence intensity is accomplished in real-time PCR software by dividing the emission intensity of the reporter dye by the emission intensity of a reference dye. In contrast to these real-time PCR systems, <a href="/en-us/category/real-time-pcr-detection-systems">Bio-Rad real-time PCR detection systems</a> utilize solid-state optical technology with long-lasting light-emitting diodes (LEDs) for fluorescence excitation and photodiodes for detection. At every position and on every scan, the optics shuttle is reproducibly centered above each well, so the light path is always identical for all wells.</p> <p>ROX (carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR systems to normalize for differences in fluorescence levels that can occur due mainly to optical path variations among wells. ROX is not involved in the PCR reaction, and ROX fluorescence levels have no relationship to the quantity of DNA in each well, so the addition of this fluorophore to a supermix provides a constant fluorescent signal during the amplification cycles. The constancy of ROX fluorescence relative to reporter fluorescence signal is used for well-to-well fluorescence normalization.</p> <p>Different types of real-time PCR systems that require a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system, particularly the type of excitation source and optics used. For some PCR master mixes, the user must determine the correct ROX concentration to optimize real-time PCR results; however, using Bio-Rad universal real-time PCR supermixes, there is no guesswork &mdash;&nbsp; our universal reagents can be used on any real-time PCR system.</p> <p>To normalize real-time PCR data, the fluorescence emission intensity of the reporter dye (for example, SYBR<sup>&reg;</sup> Green) is divided by the fluorescence emission intensity of the ROX passive reference dye. This ratio is the normalized reporter intensity, or Rn. Table 1 shows the calculation of Rn for two example wells highlighted in Figure 1. To further normalize the data, Rn+ and Rn- are calculated. Rn+ is the Rn of reactions that contain all components of the reaction including the template. Rn- is the baseline fluorescence, which is derived from the early cycles before there is a significant increase in reporter fluorescence. To determine the reporter fluorescence above baseline, &Delta;Rn is calculated as (Rn+) &ndash; (Rn-) to increase the accuracy of the fluorescence measurements.</p> <p class="caption"><strong>Table 1. Calculation of Rn for two wells at different positions.</strong> Fluorescence variation is corrected by normalizing the fluorescent reporter signal to the fluorescent signal of a passive reference dye. RFU, relative fluorescence units.</p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td colspan="4" valign="top"><span style="margin-left:80px;"><strong>Fluorescence Intensity, RFU</strong></span></td> </tr> <tr> <td valign="top"><strong>Well</strong></td> <td align="center" valign="top">Reporter</td> <td align="center" valign="top">Passive Reference</td> <td align="center" valign="top">Rn</td> </tr> <tr> <td valign="top"><strong>Center</strong></td> <td align="center" valign="top">1,500</td> <td align="center" valign="top">750</td> <td align="center" valign="top">2</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><strong>Perimeter</strong></td> <td align="center" valign="top">1,000</td> <td align="center" valign="top">500</td> <td align="center" valign="top">2</td> </tr> </tbody> </table> <p>To determine the initial concentration of input DNA, a threshold is chosen from the &Delta;Rn amplification plot that represents a detectable signal from product, and is on the exponential part of the curve. Where the curve crosses the threshold is known as the quantification cycle, or Cq.</p> Using ROX to Correct for Differences in Optical Paths between Wells <p>Figure 2 demonstrates the changes that occur with data normalization. The curves in the unnormalized data without ROX (A) are affected by their relative well positions. The amplification traces are not uniform, as ROX correction has not been applied. The Rn values are then baseline corrected by calculating &Delta;Rn (B).&nbsp; <br /> <br /> <img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/delta-rn-vs-cycle-without-rox-normalization.jpg" alt="&Delta;Rn vs. cycle without ROX normalization" width="575" height="285" /></p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/baseline-corrected-delta-rn-values-with-rox.jpg" alt="baseline-corrected &Delta;Rn values with ROX" width="575" height="285" /></p> <p class="caption"><strong>Fig. 2. Passive reference dye correction for differences in optical paths between wells. A</strong>, &Delta;Rn vs. cycle without ROX normalization; <strong>B</strong>, baseline-corrected &Delta;Rn values with ROX.</p> <p><strong>Does the Use of ROX Compensate for Differences in Well Volume?</strong> <br /> Normalization of real-time PCR data using ROX or other passive reference dye also reduces, to a small extent, the effects on data of pipetting or other volume errors.&nbsp;</p> <p>Figure 3 displays the results of a trial in which the effects of pipetting error were mimicked by varying the reaction volumes from 12 to 17 &micro;l (n = 8) while holding the amount of input template constant at 25 ng cDNA. ROX normalization decreased the standard deviation. Additionally, the Cq values were decreased by as much as 1.6 cycles for a 15 &micro;l reaction (Jordan and Kurtz 2010).&nbsp;</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/results-on-abi-7500-system-for-reactions-with-same-amount-input-template-increasing-final-reaction-volumes.jpg" alt="Results on the ABI 7500 system for reactions with the same amount of input template and increasing final reaction volumes." width="509" height="222" /></p> <p class="caption"><strong>Fig. 3. Results on the ABI 7500 system for reactions with the same amount of input template and increasing final reaction volumes.</strong> Average Cq values were measured for eight replicate reactions with identical input template amount (25 ng cDNA) and variable reaction volumes (12&ndash;17 &mu;l). ROX normalization (<img style="width: 8px; height: 8px; background-color: #a5a5a5;" src="/webroot/web/images/1x1.gif" alt="" />) lowered the standard deviations of Cq values for each replicate reaction set compared to Cq values for the identical reaction sets without ROX normalization (<img style="width: 8px; height: 8px; background-color: #00a650;" src="/webroot/web/images/1x1.gif" alt="" />). Cq, quantification cycle.</p> <p>The data in Figure 3 were obtained using an Applied Biosystems 7500 system, which is a lamp-based system. Bio-Rad real-time PCR systems use an optical shuttle, resulting in an identical light path for each well. The same protocol for changes in sample volume was repeated using the CFX96&trade; real-time PCR system. The results, shown in Figure 4, demonstrate that the optical shuttle gives more precise data with lower standard deviations. However, for both instruments, although the change in volume was as much as 40% between reactions, the volume change had no significant impact on the results, due to ROX normalization.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/results-on-cfx96-system-for-reactions-with-same-amount-input-template-increasing-final-reaction-volumes.jpg" alt="Results on the CFX96 system for reactions with the same amount of input template and increasing final reaction volumes." width="509" height="222" /></p> <p class="caption"><strong>Fig. 4. Results on the CFX96 system for reactions with the same amount of input template and increasing final reaction volumes.</strong> Average Cq values were measured for eight replicate reactions with identical input template amount (25 ng cDNA) and variable reaction volumes (12&ndash;17 &micro;l). Cq, quantification cycle.</p> <p>The addition of ROX to real-time PCR reactions can increase accuracy and reproducibility on ROX-dependent real-time PCR systems. ROX normalization can be used to compensate for spurious interwell variations in fluorescence intensity due to differences in the length of optical path, minor changes in well volume due to pipetting or other errors, and some effects of bubbles in wells. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, our universal real-time PCR supermixes perform equally well in both ROX-dependent and ROX-independent instruments.</p> Citations <p>Jordan L and Kurtz R (2010). Optical design of CFX96&trade; real-time PCR detection system eliminates the requirement of a passive reference dye. <a href="/webroot/web/pdf/lsr/literature/Bulletin_6047A.pdf" target="_blank">Bio-Rad Bulletin 6047</a>.</p> <div class="videowrap"> <div class="videoImg"><a title="Understanding Real-Time PCR Supermixes" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/supermixes_tutorial.html' );" href="javascript:void(0);"> <img style="border:none" src="/webroot/web/images/lsr/products/amplification_pcr/category_overlay_icons/global/supermixes_tutorial_thumb.jpg" alt="Understanding Real-Time PCR Supermixes" /></a></div> <div class="videoDesc"><a title="Understanding Real-Time PCR Supermixes" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/supermixes_tutorial.html' );" href="javascript:void(0);">Understanding Real-Time PCR Supermixes</a><br />Watch this short video to find out what factors affect real-time PCR results: advanced enzymes and buffer formulations provide superior inhibitor tolerance, increased processivity, and greater speed without affecting qPCR sensitivity, efficiency, or reproducibility. Learn how to compare your current master mix to a Bio-Rad supermix.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="Understanding Real-Time PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/understanding-real-time-pcr-tutorial.html' );" href="javascript:void(0);"> <img style="border:none" src="/webroot/web/images/lsr/products/amplification_pcr/category_overlay_icons/global/understanding-real-rime-pcr-trainig-module-thumb.jpg" alt="Understanding Real-Time PCR" /></a></div> <div class="videoDesc"><a title="Understanding Real-Time PCR " onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/understanding-real-time-pcr-tutorial.html' );" href="javascript:void(0);">Understanding Real-Time PCR</a><br />This video explains the principles behind the polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR), a technique using fluorescent detection of PCR products in real time to track exponential DNA amplification by PCR coupled with analytic methods to calculate the initial number of copies of a target DNA sequence with high sensitivity and accuracy.</div> <div class="clear">&nbsp;</div> </div> Why-is-my-supermix-p_1384537055 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Why-is-my-supermix-p_1384537055.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Why-is-my-supermix-p_1384537055.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Why-is-my-supermix-p_1384537055.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 09:35 AM en_US jbza3lm5 2017-11-14 06:40:18 true true Why-is-my-supermix-p_1384537055 internet/product_FAQ LSR N en 11/15/23 09:37 AM 1515436303895 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 2017-11-14 06:40:18 faq/Why-is-my-supermix-p_1384537055/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Why-is-my-supermix-p_1384537055 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Why is my supermix pink? <p>SsoAdvanced&trade; universal supermixes and iTaq&trade; universal one-step kits include our latest universal passive reference dye, which is naturally pink in color. Original iTaq universal supermixes &mdash; iTaq universal SYBR<sup>&reg;</sup> Green supermix and iTaq universal probes supermix &mdash; contained our first-generation universal passive reference dye. These supermixes have been updated with the latest-generation passive reference dye to provide enhanced long-term storage stability at 4&deg;C, reducing or even eliminating the need to freeze and thaw your supermix.</p> <p>The Bio-Rad R&amp;D team validated these updated supermixes with an extensive set of qPCR assays, evaluating relative performance, PCR efficiency, dynamic range, linearity, sensitivity, and specificity to ensure that the supermixes with the new dye provided the same high performance as the original formulations.</p> <p><strong>Related FAQs</strong></p> <ul> <li><a href="/en-us/faq/Will-my-data-remain-_1384537284/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">Will my data remain the same if I switch from SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green supermix to SsoAdvanced&trade; universal SYBR<sup>&reg;</sup> Green supermix?</a></li> <li><a href="/en-us/faq/Will-I-see-a-differe_1384540949/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">Will I see a difference in my qPCR data if I change from an older real-time PCR reagent that contained a high or low ROX concentration to a Bio-Rad supermix containing a universal reference dye?</a></li> </ul> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad&rsquo;s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.</p> /webroot/web/html/lsr/tech_support_faqs why-is-my-supermix-pink-faq.html /webroot/web/html/lsr/tech_support_faqs/why-is-my-supermix-pink-faq.html Karen Moss Why is my supermix pink? 11/15/13 09:35 AM 11/15/23 09:37 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 N 0 Which-real-time-PCR-_1384536897 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Which-real-time-PCR-_1384536897.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Which-real-time-PCR-_1384536897.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Which-real-time-PCR-_1384536897.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 09:30 AM en_US jbza3lec 2017-11-14 06:40:18 true true Which-real-time-PCR-_1384536897 internet/product_FAQ LSR N en 11/15/23 09:34 AM 1515436303852 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 2017-11-14 06:40:18 faq/Which-real-time-PCR-_1384536897/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Which-real-time-PCR-_1384536897 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Which real-time PCR supermixes and one-step RT-PCR kits contain ROX universal passive reference dye? <p>The following products contain ROX universal passive reference dye:</p> <p><strong>Supermixes for PCR and Real-Time PCR</strong></p> <ul> <li> <a href="/en-us/product/itaq-universal-supermixes/itaq-universal-sybr-green-supermix">iTaq&trade; Universal SYBR<sup>&reg;</sup> Green Supermix</a></li> <li><a href="/en-us/product/itaq-universal-supermixes/itaq-universal-probes-supermix">iTaq Universal Probes Supermix</a></li> <li><a href="http://www.bio-rad.com/en-us/product/ssoadvanced-universal-supermixes/ssoadvanced-universal-sybr-green-supermix">SsoAdvanced&trade; Universal SYBR<sup>&reg;</sup> Green Supermix</a></li> <li><a href="/en-us/product/ssoadvanced-universal-probes-supermix">SsoAdvanced Universal Probes Supermix</a></li> </ul> <p><strong>One-Step RT-PCR Kits</strong></p> <ul> <li> <a href="/en-us/product/itaq-universal-sybr-green-one-step-kit">iTaq Universal SYBR<sup>&reg;</sup> Green One-Step Kit</a></li> <li><a href="/en-us/product/itaq-universal-probes-one-step-kit">iTaq Universal Probes One-Step Kit</a></li> </ul> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad&rsquo;s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes</a> tutorial to learn more about qPCR data analysis and how to compare supermix performance.</p> /webroot/web/html/lsr/tech_support_faqs rt-pcr-supermixes-one-step-rt-pcr-reagents-contain-rox-universal-passive-reference-dye.html /webroot/web/html/lsr/tech_support_faqs/rt-pcr-supermixes-one-step-rt-pcr-reagents-contain-rox-universal-passive-reference-dye.html Karen Moss rt-pcr-supermixes-one-step-rt-pcr-reagents-contain-rox-universal-passive-reference-dye.html 11/15/13 09:30 AM 11/15/23 09:34 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 N 0 Why-were-Bio-Rad-rea_1384537662 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Why-were-Bio-Rad-rea_1384537662.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Why-were-Bio-Rad-rea_1384537662.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Why-were-Bio-Rad-rea_1384537662.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 09:41 AM en_US jbza3lko 2017-11-14 06:40:18 true true Why-were-Bio-Rad-rea_1384537662 internet/product_FAQ LSR N en 11/15/23 09:45 AM 1515436303939 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 2017-11-14 06:40:18 faq/Why-were-Bio-Rad-rea_1384537662/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Why-were-Bio-Rad-rea_1384537662 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Why were Bio-Rad real-time PCR supermixes updated with a new ROX dye? <p>The supermixes were updated with a latest-generation ROX reference dye that is naturally pink in color. This universal reference dye enhances long-term storage stability at 4&deg;C, reducing or even eliminating the need to freeze and thaw your supermix.</p> <p>The Bio-Rad R&amp;D team validated these updated supermixes with an extensive set of qPCR assays, evaluating relative performance, PCR efficiency, dynamic range, linearity, sensitivity, and specificity to ensure that the supermixes with the new dye provided the same high performance as the original formulations.</p> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad&rsquo;s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.</p> /webroot/web/html/lsr/tech_support_faqs why-were-bio-rad-real-time-pcr-supermixes-updated-with-new-rox-dye.html /webroot/web/html/lsr/tech_support_faqs/why-were-bio-rad-real-time-pcr-supermixes-updated-with-new-rox-dye.html Karen Moss Why were Bio-Rad real-time PCR supermixes updated with a new ROX dye? 11/15/13 09:41 AM 11/15/23 09:45 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 N 0 Are-there-any-perfor_1384537986 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Are-there-any-perfor_1384537986.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Are-there-any-perfor_1384537986.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Are-there-any-perfor_1384537986.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 09:49 AM en_US jbza3lkf 2017-11-14 06:40:17 true true Are-there-any-perfor_1384537986 internet/product_FAQ LSR N en 11/15/23 09:52 AM 1515436289361 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 2017-11-14 06:40:17 faq/Are-there-any-perfor_1384537986/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Are-there-any-perfor_1384537986 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Are there any performance changes in real-time PCR supermixes updated with the new ROX dye? <p>The Bio-Rad R&amp;D team validated these updated supermixes with an extensive set of qPCR assays, evaluating relative performance, PCR efficiency, dynamic range, linearity, sensitivity, and specificity to ensure that the supermixes with the new dye provided the same high performance as the original formulations. Although the vast majority of data, in terms of absolute Cq values, were identical, we noted a few assays that demonstrated Cq shifts &plusmn;0.5. This shift does not impact qPCR assay results in any manner, as Cq values in and of themselves are meaningless values.</p> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad&rsquo;s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.</p> /webroot/web/html/lsr/tech_support_faqs are-there-any-performance-changes-in-rt-pcr-supermixes-updated-with-new-rox-dye.html /webroot/web/html/lsr/tech_support_faqs/are-there-any-performance-changes-in-rt-pcr-supermixes-updated-with-new-rox-dye.html Karen Moss Are there any performance changes in real-time PCR supermixes updated with the new ROX dye? 11/15/13 09:49 AM 11/15/23 09:52 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 N 0 Will-my-data-remain-_1384537284 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Will-my-data-remain-_1384537284.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Will-my-data-remain-_1384537284.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Will-my-data-remain-_1384537284.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 09:38 AM en_US jbza3lak 2017-11-14 06:40:18 true true Will-my-data-remain-_1384537284 internet/product_FAQ LSR N en 11/15/23 09:40 AM 1515436304027 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 2017-11-14 06:40:18 faq/Will-my-data-remain-_1384537284/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Will-my-data-remain-_1384537284 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Will my data remain the same if I switch from SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green supermix to SsoAdvanced&trade; universal SYBR<sup>&reg;</sup> Green supermix? <p>An integral requirement of the reformulation was to ensure that no significant changes were noted in performance. The Bio-Rad R&amp;D team validated these updated supermixes with an extensive set of qPCR assays to assess the following attributes:</p> <ul> <li>Consistent assay results for relative expression levels</li> <li>PCR efficiency</li> <li>Linearity </li> <li>Dynamic range</li> <li>Sensitivity</li> <li>Specificity </li> </ul> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/ssoadvanced-sybr-green-supermix-compared-with-ssoadvanced-universal-sybr-green-supermix.jpg" alt="SsoAdvanced SYBR Green supermix compared with SsoAdvanced universal SYBR Green supermix" width="478" height="250" /></p> <p class="caption">SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green supermix compared with SsoAdvanced&trade; universal SYBR<sup>&reg;</sup> Green supermix. Can&rsquo;t tell which one is which? That&rsquo;s good.</p> <p>The same validation process was used in the reformulation of SsoFast&trade; probes supermix to create SsoAdvanced universal probes supermix.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/ssofast-probes-supermix-compared-with-ssoadvanced-universal-probes-supermix.jpg" alt="SsoFast probes supermix compared with SsoAdvanced universal probes supermix." width="597" height="272" /></p> <p class="caption">SsoFast probes supermix compared with SsoAdvanced universal probes supermix.</p> <p>In terms of absolute Cq values, although the vast majority of data were identical to those obtained with the previous formulation, we noted a few assays that demonstrated Cq shifts of &plusmn;0.5. This shift does not impact qPCR data in any manner, as Cq values in and of themselves are meaningless values.</p> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad&rsquo;s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.</p> /webroot/web/html/lsr/tech_support_faqs will-data-remain-same-if-switch-from-ssoadvanced-sybr-green-supermix-to-ssoadvanced-universal-sybr-green-supermix.html /webroot/web/html/lsr/tech_support_faqs/will-data-remain-same-if-switch-from-ssoadvanced-sybr-green-supermix-to-ssoadvanced-universal-sybr-green-supermix.html Karen Moss Will my data remain the same if I switch from SsoAdvanced SYBR Green supermix to SsoAdvanced univ 11/15/13 09:38 AM 11/15/23 09:40 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 N 0 Will-I-see-a-differe_1384540949 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Will-I-see-a-differe_1384540949.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Will-I-see-a-differe_1384540949.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Will-I-see-a-differe_1384540949.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 10:29 AM en_US jbza3li5 2017-11-14 06:40:18 true true Will-I-see-a-differe_1384540949 internet/product_FAQ LSR N en 11/15/23 10:41 AM 1515436303983 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 2017-11-14 06:40:18 faq/Will-I-see-a-differe_1384540949/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Will-I-see-a-differe_1384540949 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Will I see a difference in my qPCR data if I change from an older real-time PCR reagent that contained a high or low ROX concentration to a Bio-Rad supermix containing a universal reference dye? <p>Users of Bio-Rad real-time PCR detection systems will not see any significant differences in their qPCR data because the new supermix formulations simply incorporate a universal passive reference dye, which is ignored by our optical detection systems. Users of ROX-dependent real-time PCR systems (for example, Life Technologies' systems) will now be able to use any of our universal real-time PCR reagents without having to determine which product to use.</p> <p>If you were using an older product with ROX, you may see a slight shift in Cq values of less than &plusmn;0.5. However, since all the Cq values are shifted by the same amount, the relative quantitation data remain the same, with no impact on the final results.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/comparison-of-universal-reference-dye-performance-on-life-technologies-ab7900-using-actb-cdna-assay.jpg" alt="Comparison of Universal Reference Dye Performance on a Life Technologies AB7900 using an ACTB cDNA assay" width="597" height="265" /></p> <p class="caption"><strong>Comparison of universal reference dye performance on a Life Technologies AB7900 system using an ACTB cDNA assay. </strong>Amplification traces for<strong> </strong>iTaq&trade; SYBR<sup>&reg;</sup> Green supermix with ROX (<img style="width: 20px; height: 3px; background-color: #2dc489;" src="/webroot/web/images/1x1.gif" alt="" />) and iTaq&trade; universal SYBR<sup>&reg;</sup> Green supermix with the new universal reference dye (<img style="width: 20px; height: 3px; background-color: #564e8a;" src="/webroot/web/images/1x1.gif" alt="" />). An earlier Cq shift of &lt;0.3 is observed with the new universal dye.</p> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad&rsquo;s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.&nbsp;</p> /webroot/web/html/lsr/tech_support_faqs will-I-see-difference-in-qpcr-data-if-change-from-older-rtpcr-reagent-to-biorad-supermix-with-universal-reference-dye.html /webroot/web/html/lsr/tech_support_faqs/will-I-see-difference-in-qpcr-data-if-change-from-older-rtpcr-reagent-to-biorad-supermix-with-universal-reference-dye.html Karen Moss Will I see a difference in my qPCR data Will I see a difference in my qPCR data if I change from an older real-time PCR reagent that contained a high or low ROX concentration to a Bio-Rad supermix containing a universal reference dye? 11/15/13 10:29 AM 11/15/23 10:41 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 N 0 Do-levels-of-RFUs-fo_1384541547 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Do-levels-of-RFUs-fo_1384541547.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Do-levels-of-RFUs-fo_1384541547.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Do-levels-of-RFUs-fo_1384541547.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 10:46 AM en_US jbza3lji 2017-11-14 06:40:17 true true Do-levels-of-RFUs-fo_1384541547 internet/product_FAQ LSR N en 11/15/23 10:51 AM 1515436290177 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 2017-11-14 06:40:17 faq/Do-levels-of-RFUs-fo_1384541547/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Do-levels-of-RFUs-fo_1384541547 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Do the levels of relative fluorescence units (RFUs) for a qPCR reaction have an impact on my data? <p>No. The Cq values will remain the same regardless of the RFU level. The reporter dye is simply used to yield a signal in the exponential phase of PCR so that a Cq value can be derived. The absolute magnitude of the fluorescence level is thus unimportant.</p> <p>The RFU signal is determined by several factors including</p> <ul> <li>The reporter dye used &mdash; green dyes such as FAM and SYBR<sup>&reg;</sup> are brighter than Cy5 and other dyes closer to the red end of the spectrum</li> <li>Reagent formulation &mdash; components such as buffers and salts affect pH and ionic strength, which in turn affect the RFU; thus, RFU levels may vary among real-time PCR supermixes from different vendors</li> </ul> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/comparison-of-universal-reference-dye-performance-on-life-technologies-ab7900-using-mmp9-cdna-assay.jpg" alt="Comparison of Universal Reference Dye Performance on a Life Technologies AB7900 using an MMP9 cDNA assay" width="597" height="275" /></p> <p class="caption"><strong>Comparison of universal reference dye performance on a Life Technologies AB7900 system using an MMP9 cDNA assay.</strong> Amplification traces for<strong> </strong>iTaq&trade; SYBR<sup>&reg;</sup> Green supermix with ROX (<img style="width: 20px; height: 3px; background-color: #2dc489;" src="/webroot/web/images/1x1.gif" alt="" />) and iTaq&trade; universal SYBR<sup>&reg;</sup> Green supermix with the new universal reference dye <span class="forcebreaks"> (<img style="width: 20px; height: 3px; background-color: #564e8a;" src="/webroot/web/images/1x1.gif" alt="" />).</span> No Cq shift was observed in the data.</p> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, Bio-Rad&rsquo;s universal real-time PCR supermixes are designed for high performance on all real-time PCR detection systems, both systems requiring ROX normalization and ROX-independent systems.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.&nbsp;</p> /webroot/web/html/lsr/tech_support_faqs do-levels-of-rfus-for-qPCR-reaction-have-impact-on-data.html /webroot/web/html/lsr/tech_support_faqs/do-levels-of-rfus-for-qPCR-reaction-have-impact-on-data.html Karen Moss Do the levels of relative fluorescence units (RFUs) for a qPCR reaction have an impact on my data? 11/15/13 10:46 AM 11/15/23 10:51 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 N 0 What-components-are-_1384541752 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/What-components-are-_1384541752.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/What-components-are-_1384541752.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/What-components-are-_1384541752.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 10:53 AM en_US jbza3lk6 2017-11-14 06:40:18 true true What-components-are-_1384541752 internet/product_FAQ LSR N en 11/15/23 10:55 AM 1515436303763 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 2017-11-14 06:40:18 faq/What-components-are-_1384541752/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye What-components-are-_1384541752 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye What components are in Bio-Rad real-time PCR supermixes? <p>In addition to the standard PCR constituents, the advanced components in our supermixes include specially engineered polymerases as well as surfactants, optimized buffers, stabilizers, and other proprietary compounds designed to increase stability and efficiency.</p> <p>Bio-Rad real-time PCR detection systems do not require ROX normalization, but our universal real-time PCR supermixes contain a latest-generation ROX reference dye to perform equally well on both ROX-dependent and ROX-independent real-time PCR detection systems.</p> <p><a href="/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">See Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye</a> in our Applications and Technologies pages to learn why passive reference dyes are required for data normalization in some real-time PCR systems and not others.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.&nbsp;</p> /webroot/web/html/lsr/tech_support_faqs what-components-are-in-bio-rad-real-time-pcr-supermixes.html /webroot/web/html/lsr/tech_support_faqs/what-components-are-in-bio-rad-real-time-pcr-supermixes.html Karen Moss What components are in Bio-Rad real-time PCR supermixes? 11/15/13 10:53 AM 11/15/23 10:55 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 N 0 What-is-the-Sso7d-fu_1384542041 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/What-is-the-Sso7d-fu_1384542041.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/What-is-the-Sso7d-fu_1384542041.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/What-is-the-Sso7d-fu_1384542041.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 10:56 AM en_US jbza3l5z 2017-11-14 06:40:18 true true What-is-the-Sso7d-fu_1384542041 internet/product_FAQ LSR N en 11/15/23 10:58 AM 1515436303808 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 2017-11-14 06:40:18 faq/What-is-the-Sso7d-fu_1384542041/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye What-is-the-Sso7d-fu_1384542041 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye What is the Sso7d fusion polymerase? <p>Bio-Rad Laboratories&rsquo; patented Sso7d fusion enzyme is a fast, high-fidelity polymerase coupled to a double-stranded-DNA binding protein that stabilizes the polymerase-template complex for increased processivity.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.&nbsp;</p> /webroot/web/html/lsr/tech_support_faqs what-is-the-sso7d-fusion-polymerase.html /webroot/web/html/lsr/tech_support_faqs/what-is-the-sso7d-fusion-polymerase.html Karen Moss What is the Sso7d fusion polymerase? 11/15/13 10:56 AM 11/15/23 10:58 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 N 0 How-will-Sso7d-fusio_1384542457 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/How-will-Sso7d-fusio_1384542457.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/How-will-Sso7d-fusio_1384542457.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/How-will-Sso7d-fusio_1384542457.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 11:01 AM en_US jbza3l7a 2017-11-14 06:40:18 true true How-will-Sso7d-fusio_1384542457 internet/product_FAQ LSR N en 11/15/23 11:06 AM 1515436291894 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 2017-11-14 06:40:18 faq/How-will-Sso7d-fusio_1384542457/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye How-will-Sso7d-fusio_1384542457 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye How will Sso7d fusion polymerase improve my qPCR assays? <p>By stabilizing the polymerase-template complex, Sso7d fusion polymerase provides a number of advantages over ordinary DNA polymerases. The tighter association between the enzyme and the template strand results in increased processivity, increased read-through in regions of GC-rich sequence or strong secondary structure, longer products, and higher yields; additionally Sso7d fusion polymerase displays superior tolerance to PCR inhibitors.</p> <p><a href="/supermixes_tutorial">View our Understanding Real-Time Supermixes tutorial</a> to learn more about qPCR data analysis and how to compare supermix performance.</p> /webroot/web/html/lsr/tech_support_faqs how-will-ssso7d-fusion-polymerase-improve-my-qpcr-assays.html /webroot/web/html/lsr/tech_support_faqs/how-will-ssso7d-fusion-polymerase-improve-my-qpcr-assays.html Karen Moss How will Sso7d fusion polymerase improve my qPCR assays? 11/15/13 11:01 AM 11/15/23 11:06 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 N 0 Is-there-a-list-of-e_1384542839 <?xml version="1.0" encoding="UTF-8"?> <Deployments> <Node Name="QA"> <Deployment Id="StandardMetadata.jbza3lmrjbza3lms" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3lmrjbza3lms"> <Name>StandardMetadata</Name> <TargetEnviroment>QA</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Is-there-a-list-of-e_1384542839.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="7" DayOfWeek="7" Year="2018" Hour="17" Minute="50" Second="40" ZoneOffsetMillis="0" Time="1515347440361">Sun Jan 07 17:50:40 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODWEST"> <Deployment Id="StandardMetadata.jbza3midjbza3mie" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3mie"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODWEST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Is-there-a-list-of-e_1384542839.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="17" Minute="28" Second="24" ZoneOffsetMillis="0" Time="1515432504036">Mon Jan 08 17:28:24 UTC 2018</LastExecutionTime> </Node> <Node Name="PRODEAST"> <Deployment Id="StandardMetadata.jbza3midjbza3miu" Class="com.interwoven.livesite.lscs.model.LSCSDeploymentImpl" Instance="jbza3midjbza3miu"> <Name>StandardMetadata</Name> <TargetEnviroment>PRODEAST</TargetEnviroment> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Deployment> <Vpath>/default/main/bio-rad/WORKAREA/default/templatedata/internet/product_FAQ/data/LSR/Is-there-a-list-of-e_1384542839.xml</Vpath> <LastExecutionTime Class="java.util.Date" Month="1" DayOfMonth="8" DayOfWeek="1" Year="2018" Hour="18" Minute="9" Second="12" ZoneOffsetMillis="0" Time="1515434952721">Mon Jan 08 18:09:12 UTC 2018</LastExecutionTime> </Node> </Deployments> 11/15/13 11:10 AM en_US jbza3lmh 2017-11-14 06:40:18 true true Is-there-a-list-of-e_1384542839 internet/product_FAQ LSR N en 11/15/23 11:13 AM 1515436292921 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 2017-11-14 06:40:18 faq/Is-there-a-list-of-e_1384542839/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye Is-there-a-list-of-e_1384542839 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye I understand that some Bio-Rad qPCR supermixes are being discontinued. Is there a list of equivalent replacement real-time PCR reagents? <p>Yes. To find an equivalent replacement product for a discontinued qPCR reagent, view this table cross-referencing the discontinued reagents with the new products.</p> <p><strong>Discontinued PCR Supermixes and Replacement Products</strong></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr class="pd_tableheader"> <td><strong>Discontinued Kits</strong></td> <td><strong>Catalog Number</strong></td> <td class="pd_centeredcell"><strong>Supermix Volume</strong></td> <td><strong>Replacement Kits</strong></td> <td class="pd_centeredcell"><strong>Supermix Volume</strong></td> </tr> <tr class="pd_colorbackground" valign="top"> <td>SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green Supermix</td> <td>172-5260</td> <td class="pd_centeredcell">2 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5270">SsoAdvanced&trade; Universal SYBR<sup>&reg;</sup> Green Supermix (172-5270)</a></td> <td class="pd_centeredcell">2 ml</td> </tr> <tr valign="top"> <td>SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green Supermix</td> <td>172-5261</td> <td class="pd_centeredcell">5 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5271">SsoAdvanced&trade; Universal SYBR<sup>&reg;</sup> Green Supermix (172-5271)</a></td> <td class="pd_centeredcell">5 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td>SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green Supermix</td> <td>172-5262</td> <td class="pd_centeredcell">10 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5272">SsoAdvanced&trade; Universal SYBR<sup>&reg;</sup> Green Supermix (172-5272)</a></td> <td class="pd_centeredcell">10 ml</td> </tr> <tr valign="top"> <td>SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green Supermix</td> <td>172-5264</td> <td class="pd_centeredcell">25 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5274">SsoAdvanced&trade; Universal SYBR<sup>&reg;</sup> Green Supermix (172-5274)</a></td> <td class="pd_centeredcell">25 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td>SsoAdvanced&trade; SYBR<sup>&reg;</sup> Green Supermix</td> <td>172-5265</td> <td class="pd_centeredcell">50 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5275">SsoAdvanced&trade; Universal SYBR<sup>&reg;</sup> Green Supermix (172-5275)</a></td> <td class="pd_centeredcell">50 ml</td> </tr> <tr valign="top"> <td>SsoFast&trade; Probes Supermix&nbsp;</td> <td>172-5230</td> <td class="pd_centeredcell">2 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5280">SsoAdvanced&trade; Universal Probes Supermix (172-5280)</a></td> <td class="pd_centeredcell">2 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td>SsoFast Probes Supermix&nbsp;</td> <td>172-5231</td> <td class="pd_centeredcell">5 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5281">SsoAdvanced&trade; Universal Probes Supermix (172-5281)</a></td> <td class="pd_centeredcell">5 ml</td> </tr> <tr valign="top"> <td>SsoFast Probes Supermix&nbsp;</td> <td>172-5232</td> <td class="pd_centeredcell">10 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5282">SsoAdvanced&trade; Universal Probes Supermix (172-5282)</a></td> <td class="pd_centeredcell">10 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td rowspan="2">SsoFast Probes Supermix&nbsp;</td> <td rowspan="2">172-5233</td> <td class="pd_centeredcell" rowspan="2">20 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5284">SsoAdvanced&trade; Universal Probes Supermix (172-5284)</a></td> <td class="pd_centeredcell">25 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5285">SsoAdvanced&trade; Universal Probes Supermix (172-5285)</a></td> <td class="pd_centeredcell">50 ml</td> </tr> <tr valign="top"> <td>SsoFast Probes Supermix with ROX</td> <td>172-5250</td> <td class="pd_centeredcell">2 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5280">SsoAdvanced&trade; Universal Probes Supermix (172-5280)</a></td> <td class="pd_centeredcell">2 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td>SsoFast Probes Supermix with ROX</td> <td>172-5251</td> <td class="pd_centeredcell">5 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5281">SsoAdvanced&trade; Universal Probes Supermix (172-5281)</a></td> <td class="pd_centeredcell">5 ml</td> </tr> <tr valign="top"> <td>SsoFast Probes Supermix with ROX</td> <td>172-5252</td> <td class="pd_centeredcell">10 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5282">SsoAdvanced&trade; Universal Probes Supermix (172-5282)</a></td> <td class="pd_centeredcell">10 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td rowspan="2">SsoFast Probes Supermix with ROX</td> <td rowspan="2">172-5253</td> <td class="pd_centeredcell" rowspan="2">20 ml</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5284">SsoAdvanced&trade; Universal Probes Supermix (172-5284)</a></td> <td class="pd_centeredcell">25 ml</td> </tr> <tr class="pd_colorbackground" valign="top"> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5285">SsoAdvanced&trade; Universal Probes Supermix (172-5285)</a></td> <td class="pd_centeredcell">50 ml</td> </tr> <tr valign="top"> <td>iScript&trade; One-Step RT-PCR Kit with SYBR<sup>&reg;</sup> Green</td> <td>170-8892</td> <td class="pd_centeredcell">50 reactions</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5150">iTaq&trade; Universal SYBR<sup>&reg;</sup> Green One-Step Kit (172-5150)</a></td> <td class="pd_centeredcell">100 reactions</td> </tr> <tr class="pd_colorbackground" valign="top"> <td>iScript&trade; One-Step RT-PCR Kit with SYBR<sup>&reg;</sup> Green</td> <td>170-8893</td> <td class="pd_centeredcell">200 reactions</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5151">iTaq&trade; Universal SYBR<sup>&reg;</sup> Green One-Step Kit (172-5151)</a></td> <td class="pd_centeredcell">500 reactions</td> </tr> <tr valign="top"> <td>iScript&trade; One-Step RT-PCR Kit for Probes</td> <td>170-8894</td> <td class="pd_centeredcell">50 reactions</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5140">iTaq&trade; Universal Probes One-Step Kit (172-5140)</a></td> <td class="pd_centeredcell">100 reactions</td> </tr> <tr class="pd_colorbackground" valign="top"> <td>iScript One-Step RT-PCR Kit for Probes</td> <td>170-8895</td> <td class="pd_centeredcell">200 reactions</td> <td><a href="/evportal/destination/commerce/sku_detail?cmd=catProductDetail&amp;vertical=LSR&amp;productID=172-5141">iTaq&trade; Universal Probes One-Step Kit (172-5141)</a></td> <td class="pd_centeredcell">500 reactions</td> </tr> </tbody> </table> /webroot/web/html/lsr/tech_support_faqs is-there-a-list-of-equivalent-replacement-real-time-pcr-reagents.html /webroot/web/html/lsr/tech_support_faqs/is-there-a-list-of-equivalent-replacement-real-time-pcr-reagents.html Karen Moss Is there a list of equivalent replacement real-time PCR reagents? 11/15/13 11:10 AM 11/15/23 11:13 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 N 0 Karen Moss Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye 11/13/13 05:17 PM 11/13/23 05:32 PM 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 qPCR/Real-Time PCR Reagents /en-us/applications-technologies/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye?ID=LUSO6XKSY

Certain real-time PCR instruments require an internal reference dye for fluorescent signal normalization and correction of well-to-well optical variations. Variation in fluorescence can result from instrument design. ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization throughout the real-time qPCR assay.

Related Topics: qPCR Instrumentation, qPCR/Real-Time PCR Reagents, and qPCR Assay Design and Optimization.


 

Understanding Cq Values, Optical Systems, and Passive Reference Dyes

A Cq (quantification cycle) value is an exponential number derived from the intersection of the threshold and a given amplification curve and is a relative concentration measurement of the target presence in the qPCR reaction. Several factors independent of the sample influence the Cq value — the optical system, PCR supermix, threshold and baseline settings, and the presence or absence of a passive reference dye such as ROX. Before explaining how a passive reference dye like ROX impacts Cq values and qPCR data, it is important to understand how optical real-time PCR detection systems work.

The optical design of most real-time PCR systems, using a lamp or other single stationary light source, creates illumination and detection light paths that vary for each well of the thermal cycler block. The variation in light path lengths produces different absolute fluorescence measurements for wells that contain the same concentration of a reporter fluorophore. A well with a shorter light path, typically located in the middle of the block, will have a higher fluorescence reading compared to a well with a longer light path, typically located on the perimeter of the block (Figure 1).

Light path differences between wells of a lamp-based real-time PCR system

Fig. 1. Light path differences between wells of a lamp-based real-time PCR system. The well in the center of the block has a shorter light path and, subsequently, emits a higher fluorescence signal compared to the well on the perimeter, which has a longer light path.

The differences in absolute fluorescence levels do not impact real-time PCR results because results are determined by measuring the change in fluorescence of a reporter measured over the course of the reaction. For real-time PCR data processing, the sample fluorescence for each well is first baseline subtracted and then the quantification cycle (Cq) value is determined as a cycle number at which fluorescence has increased above background.

 

 

Normalization of Fluorescence Data Using ROX

Some lamp- and laser-based real-time PCR systems utilize an internal reference dye to normalize well-to-well fluorescence signal differences resulting from optical path length variations. Normalizing the fluorescence intensity is accomplished in real-time PCR software by dividing the emission intensity of the reporter dye by the emission intensity of a reference dye. In contrast to these real-time PCR systems, Bio-Rad real-time PCR detection systems utilize solid-state optical technology with long-lasting light-emitting diodes (LEDs) for fluorescence excitation and photodiodes for detection. At every position and on every scan, the optics shuttle is reproducibly centered above each well, so the light path is always identical for all wells.

ROX (carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR systems to normalize for differences in fluorescence levels that can occur due mainly to optical path variations among wells. ROX is not involved in the PCR reaction, and ROX fluorescence levels have no relationship to the quantity of DNA in each well, so the addition of this fluorophore to a supermix provides a constant fluorescent signal during the amplification cycles. The constancy of ROX fluorescence relative to reporter fluorescence signal is used for well-to-well fluorescence normalization.

Different types of real-time PCR systems that require a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system, particularly the type of excitation source and optics used. For some PCR master mixes, the user must determine the correct ROX concentration to optimize real-time PCR results; however, using Bio-Rad universal real-time PCR supermixes, there is no guesswork —  our universal reagents can be used on any real-time PCR system.

To normalize real-time PCR data, the fluorescence emission intensity of the reporter dye (for example, SYBR® Green) is divided by the fluorescence emission intensity of the ROX passive reference dye. This ratio is the normalized reporter intensity, or Rn. Table 1 shows the calculation of Rn for two example wells highlighted in Figure 1. To further normalize the data, Rn+ and Rn- are calculated. Rn+ is the Rn of reactions that contain all components of the reaction including the template. Rn- is the baseline fluorescence, which is derived from the early cycles before there is a significant increase in reporter fluorescence. To determine the reporter fluorescence above baseline, ΔRn is calculated as (Rn+) – (Rn-) to increase the accuracy of the fluorescence measurements.

Table 1. Calculation of Rn for two wells at different positions. Fluorescence variation is corrected by normalizing the fluorescent reporter signal to the fluorescent signal of a passive reference dye. RFU, relative fluorescence units.

Fluorescence Intensity, RFU
Well Reporter Passive Reference Rn
Center 1,500 750 2
Perimeter 1,000 500 2

To determine the initial concentration of input DNA, a threshold is chosen from the ΔRn amplification plot that represents a detectable signal from product, and is on the exponential part of the curve. Where the curve crosses the threshold is known as the quantification cycle, or Cq.

 

Using ROX to Correct for Differences in Optical Paths between Wells

Figure 2 demonstrates the changes that occur with data normalization. The curves in the unnormalized data without ROX (A) are affected by their relative well positions. The amplification traces are not uniform, as ROX correction has not been applied. The Rn values are then baseline corrected by calculating ΔRn (B). 

ΔRn vs. cycle without ROX normalization

baseline-corrected ΔRn values with ROX

Fig. 2. Passive reference dye correction for differences in optical paths between wells. A, ΔRn vs. cycle without ROX normalization; B, baseline-corrected ΔRn values with ROX.

Does the Use of ROX Compensate for Differences in Well Volume?
Normalization of real-time PCR data using ROX or other passive reference dye also reduces, to a small extent, the effects on data of pipetting or other volume errors. 

Figure 3 displays the results of a trial in which the effects of pipetting error were mimicked by varying the reaction volumes from 12 to 17 µl (n = 8) while holding the amount of input template constant at 25 ng cDNA. ROX normalization decreased the standard deviation. Additionally, the Cq values were decreased by as much as 1.6 cycles for a 15 µl reaction (Jordan and Kurtz 2010). 

Results on the ABI 7500 system for reactions with the same amount of input template and increasing final reaction volumes.

Fig. 3. Results on the ABI 7500 system for reactions with the same amount of input template and increasing final reaction volumes. Average Cq values were measured for eight replicate reactions with identical input template amount (25 ng cDNA) and variable reaction volumes (12–17 μl). ROX normalization () lowered the standard deviations of Cq values for each replicate reaction set compared to Cq values for the identical reaction sets without ROX normalization (). Cq, quantification cycle.

The data in Figure 3 were obtained using an Applied Biosystems 7500 system, which is a lamp-based system. Bio-Rad real-time PCR systems use an optical shuttle, resulting in an identical light path for each well. The same protocol for changes in sample volume was repeated using the CFX96™ real-time PCR system. The results, shown in Figure 4, demonstrate that the optical shuttle gives more precise data with lower standard deviations. However, for both instruments, although the change in volume was as much as 40% between reactions, the volume change had no significant impact on the results, due to ROX normalization.

Results on the CFX96 system for reactions with the same amount of input template and increasing final reaction volumes.

Fig. 4. Results on the CFX96 system for reactions with the same amount of input template and increasing final reaction volumes. Average Cq values were measured for eight replicate reactions with identical input template amount (25 ng cDNA) and variable reaction volumes (12–17 µl). Cq, quantification cycle.

The addition of ROX to real-time PCR reactions can increase accuracy and reproducibility on ROX-dependent real-time PCR systems. ROX normalization can be used to compensate for spurious interwell variations in fluorescence intensity due to differences in the length of optical path, minor changes in well volume due to pipetting or other errors, and some effects of bubbles in wells. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, our universal real-time PCR supermixes perform equally well in both ROX-dependent and ROX-independent instruments.

 

Citations

Jordan L and Kurtz R (2010). Optical design of CFX96™ real-time PCR detection system eliminates the requirement of a passive reference dye. Bio-Rad Bulletin 6047.

 

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MW472W15 [x-forwarded-proto] = [http] [accept-language] = [en-US] [x-forwarded-port] = [80] [x-forwarded-for] = [180.76.15.150, 10.232.2.51] [accept] = [*/*] [seourl] = [/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye] [x-amzn-trace-id] = [Root=1-5b4f52f3-21272b4043e05be0c71c87c0] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=MW472W15] [host] = [10.232.16.28:1776] [x-request-uri] = [/en-us/applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye] [connection] = [Keep-Alive] [accept-encoding] = [gzip] [user-agent] = [Mozilla/5.0 (compatible; Baiduspider/2.0; +http://www.baidu.com/search/spider.html)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/normalization-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye LSR MW472W15 Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye /webroot/web/html/lsr/solutions/technologies/qpcr_real-time_pcr <p>Certain real-time PCR instruments require an internal reference dye for fluorescent signal normalization and correction of well-to-well optical variations. 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ROX passive reference dye is an inert additive that provides a constant fluorescent signal for sample normalization throughout the real-time qPCR assay.</p> <p>Related Topics: <a href="/en-us/applications-technologies/qpcr-instrumentation">qPCR Instrumentation</a>, <a href="/en-us/applications-technologies/qpcr-real-time-pcr-reagents">qPCR/Real-Time PCR Reagents</a>, and <a href="/en-us/applications-technologies/qpcr-assay-design-optimization">qPCR Assay Design and Optimization</a>.</p> <br /> <!-- <div style="border: 1px solid #cccccc; width: 614px; height: 49px; margin-top: 20px; margin-bottom: 20px;"> <div style="float: left; width: 123px; height: 49px; background-color: #333333; margin-right: 10px; padding-left: 14px;"> <div style="font-size:13px; color:#FFF; margin-top: .8em;" mce_style="font-size:13px; color:#FFF; margin-top: .8em;"><b>WHY IS MY SUPERMIX PINK?</b></div> </div> <div style="margin-top: .9em; padding-right:5px" mce_style="margin-top: .9em; padding-right:5px"><b>Bio-Rad&rsquo;s universal real-time PCR supermixes include a new pink universal passive reference dye that is suitable for use on any qPCR system. <a style="font-weight:bold" mce_style="font-weight:bold" href="/en-us/faq/Why-is-my-supermix-p_1384537055/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye" mce_href="/en-us/faq/Why-is-my-supermix-p_1384537055/normalization-of-real-time-pcr-fluorescence-data-with-rox-passive-reference-dye">Find out why</a></b></div> </div> --> Understanding Cq Values, Optical Systems, and Passive Reference Dyes <p>A Cq (quantification cycle) value is an exponential number derived from the intersection of the threshold and a given amplification curve and is a relative concentration measurement of the target presence in the qPCR reaction. Several factors independent of the sample influence the Cq value &mdash; the optical system, PCR supermix, threshold and baseline settings, and the presence or absence of a passive reference dye such as ROX. Before explaining how a passive reference dye like ROX impacts Cq values and qPCR data, it is important to understand how optical real-time PCR detection systems work.</p> <p>The optical design of most real-time PCR systems, using a lamp or other single stationary light source, creates illumination and detection light paths that vary for each well of the thermal cycler block. The variation in light path lengths produces different absolute fluorescence measurements for wells that contain the same concentration of a reporter fluorophore. A well with a shorter light path, typically located in the middle of the block, will have a higher fluorescence reading compared to a well with a longer light path, typically located on the perimeter of the block (Figure 1).</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/light-path-differences-between-wells-of-a-lamp-based-real-time-pcr-system.jpg" alt="Light path differences between wells of a lamp-based real-time PCR system" width="423" height="255" /></p> <p class="caption"><strong>Fig. 1. Light path differences between wells of a lamp-based real-time PCR system.</strong> The well in the center of the block has a shorter light path and, subsequently, emits a higher fluorescence signal compared to the well on the perimeter, which has a longer light path.</p> <p>The differences in absolute fluorescence levels do not impact real-time PCR results because results are determined by measuring the change in fluorescence of a reporter measured over the course of the reaction. For real-time PCR data processing, the sample fluorescence for each well is first baseline subtracted and then the quantification cycle (Cq) value is determined as a cycle number at which fluorescence has increased above background.</p> <p>&nbsp;</p> Normalization of Fluorescence Data Using ROX <p>Some lamp- and laser-based real-time PCR systems utilize an internal reference dye to normalize well-to-well fluorescence signal differences resulting from optical path length variations. Normalizing the fluorescence intensity is accomplished in real-time PCR software by dividing the emission intensity of the reporter dye by the emission intensity of a reference dye. In contrast to these real-time PCR systems, <a href="/en-us/category/real-time-pcr-detection-systems">Bio-Rad real-time PCR detection systems</a> utilize solid-state optical technology with long-lasting light-emitting diodes (LEDs) for fluorescence excitation and photodiodes for detection. At every position and on every scan, the optics shuttle is reproducibly centered above each well, so the light path is always identical for all wells.</p> <p>ROX (carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR systems to normalize for differences in fluorescence levels that can occur due mainly to optical path variations among wells. ROX is not involved in the PCR reaction, and ROX fluorescence levels have no relationship to the quantity of DNA in each well, so the addition of this fluorophore to a supermix provides a constant fluorescent signal during the amplification cycles. The constancy of ROX fluorescence relative to reporter fluorescence signal is used for well-to-well fluorescence normalization.</p> <p>Different types of real-time PCR systems that require a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system, particularly the type of excitation source and optics used. For some PCR master mixes, the user must determine the correct ROX concentration to optimize real-time PCR results; however, using Bio-Rad universal real-time PCR supermixes, there is no guesswork &mdash;&nbsp; our universal reagents can be used on any real-time PCR system.</p> <p>To normalize real-time PCR data, the fluorescence emission intensity of the reporter dye (for example, SYBR<sup>&reg;</sup> Green) is divided by the fluorescence emission intensity of the ROX passive reference dye. This ratio is the normalized reporter intensity, or Rn. Table 1 shows the calculation of Rn for two example wells highlighted in Figure 1. To further normalize the data, Rn+ and Rn- are calculated. Rn+ is the Rn of reactions that contain all components of the reaction including the template. Rn- is the baseline fluorescence, which is derived from the early cycles before there is a significant increase in reporter fluorescence. To determine the reporter fluorescence above baseline, &Delta;Rn is calculated as (Rn+) &ndash; (Rn-) to increase the accuracy of the fluorescence measurements.</p> <p class="caption"><strong>Table 1. Calculation of Rn for two wells at different positions.</strong> Fluorescence variation is corrected by normalizing the fluorescent reporter signal to the fluorescent signal of a passive reference dye. RFU, relative fluorescence units.</p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td colspan="4" valign="top"><span style="margin-left:80px;"><strong>Fluorescence Intensity, RFU</strong></span></td> </tr> <tr> <td valign="top"><strong>Well</strong></td> <td align="center" valign="top">Reporter</td> <td align="center" valign="top">Passive Reference</td> <td align="center" valign="top">Rn</td> </tr> <tr> <td valign="top"><strong>Center</strong></td> <td align="center" valign="top">1,500</td> <td align="center" valign="top">750</td> <td align="center" valign="top">2</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><strong>Perimeter</strong></td> <td align="center" valign="top">1,000</td> <td align="center" valign="top">500</td> <td align="center" valign="top">2</td> </tr> </tbody> </table> <p>To determine the initial concentration of input DNA, a threshold is chosen from the &Delta;Rn amplification plot that represents a detectable signal from product, and is on the exponential part of the curve. Where the curve crosses the threshold is known as the quantification cycle, or Cq.</p> Using ROX to Correct for Differences in Optical Paths between Wells <p>Figure 2 demonstrates the changes that occur with data normalization. The curves in the unnormalized data without ROX (A) are affected by their relative well positions. The amplification traces are not uniform, as ROX correction has not been applied. The Rn values are then baseline corrected by calculating &Delta;Rn (B).&nbsp; <br /> <br /> <img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/delta-rn-vs-cycle-without-rox-normalization.jpg" alt="&Delta;Rn vs. cycle without ROX normalization" width="575" height="285" /></p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/baseline-corrected-delta-rn-values-with-rox.jpg" alt="baseline-corrected &Delta;Rn values with ROX" width="575" height="285" /></p> <p class="caption"><strong>Fig. 2. Passive reference dye correction for differences in optical paths between wells. A</strong>, &Delta;Rn vs. cycle without ROX normalization; <strong>B</strong>, baseline-corrected &Delta;Rn values with ROX.</p> <p><strong>Does the Use of ROX Compensate for Differences in Well Volume?</strong> <br /> Normalization of real-time PCR data using ROX or other passive reference dye also reduces, to a small extent, the effects on data of pipetting or other volume errors.&nbsp;</p> <p>Figure 3 displays the results of a trial in which the effects of pipetting error were mimicked by varying the reaction volumes from 12 to 17 &micro;l (n = 8) while holding the amount of input template constant at 25 ng cDNA. ROX normalization decreased the standard deviation. Additionally, the Cq values were decreased by as much as 1.6 cycles for a 15 &micro;l reaction (Jordan and Kurtz 2010).&nbsp;</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/results-on-abi-7500-system-for-reactions-with-same-amount-input-template-increasing-final-reaction-volumes.jpg" alt="Results on the ABI 7500 system for reactions with the same amount of input template and increasing final reaction volumes." width="509" height="222" /></p> <p class="caption"><strong>Fig. 3. Results on the ABI 7500 system for reactions with the same amount of input template and increasing final reaction volumes.</strong> Average Cq values were measured for eight replicate reactions with identical input template amount (25 ng cDNA) and variable reaction volumes (12&ndash;17 &mu;l). ROX normalization (<img style="width: 8px; height: 8px; background-color: #a5a5a5;" src="/webroot/web/images/1x1.gif" alt="" />) lowered the standard deviations of Cq values for each replicate reaction set compared to Cq values for the identical reaction sets without ROX normalization (<img style="width: 8px; height: 8px; background-color: #00a650;" src="/webroot/web/images/1x1.gif" alt="" />). Cq, quantification cycle.</p> <p>The data in Figure 3 were obtained using an Applied Biosystems 7500 system, which is a lamp-based system. Bio-Rad real-time PCR systems use an optical shuttle, resulting in an identical light path for each well. The same protocol for changes in sample volume was repeated using the CFX96&trade; real-time PCR system. The results, shown in Figure 4, demonstrate that the optical shuttle gives more precise data with lower standard deviations. However, for both instruments, although the change in volume was as much as 40% between reactions, the volume change had no significant impact on the results, due to ROX normalization.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/gene_expression/qPCR_real-time_PCR/technology_detail/results-on-cfx96-system-for-reactions-with-same-amount-input-template-increasing-final-reaction-volumes.jpg" alt="Results on the CFX96 system for reactions with the same amount of input template and increasing final reaction volumes." width="509" height="222" /></p> <p class="caption"><strong>Fig. 4. Results on the CFX96 system for reactions with the same amount of input template and increasing final reaction volumes.</strong> Average Cq values were measured for eight replicate reactions with identical input template amount (25 ng cDNA) and variable reaction volumes (12&ndash;17 &micro;l). Cq, quantification cycle.</p> <p>The addition of ROX to real-time PCR reactions can increase accuracy and reproducibility on ROX-dependent real-time PCR systems. ROX normalization can be used to compensate for spurious interwell variations in fluorescence intensity due to differences in the length of optical path, minor changes in well volume due to pipetting or other errors, and some effects of bubbles in wells. Although Bio-Rad real-time PCR detection systems do not require ROX for internal normalization, our universal real-time PCR supermixes perform equally well in both ROX-dependent and ROX-independent instruments.</p> Citations <p>Jordan L and Kurtz R (2010). Optical design of CFX96&trade; real-time PCR detection system eliminates the requirement of a passive reference dye. <a href="/webroot/web/pdf/lsr/literature/Bulletin_6047A.pdf" target="_blank">Bio-Rad Bulletin 6047</a>.</p> <div class="videowrap"> <div class="videoImg"><a title="Understanding Real-Time PCR Supermixes" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/supermixes_tutorial.html' );" href="javascript:void(0);"> <img style="border:none" src="/webroot/web/images/lsr/products/amplification_pcr/category_overlay_icons/global/supermixes_tutorial_thumb.jpg" alt="Understanding Real-Time PCR Supermixes" /></a></div> <div class="videoDesc"><a title="Understanding Real-Time PCR Supermixes" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/supermixes_tutorial.html' );" href="javascript:void(0);">Understanding Real-Time PCR Supermixes</a><br />Watch this short video to find out what factors affect real-time PCR results: advanced enzymes and buffer formulations provide superior inhibitor tolerance, increased processivity, and greater speed without affecting qPCR sensitivity, efficiency, or reproducibility. Learn how to compare your current master mix to a Bio-Rad supermix.</div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="Understanding Real-Time PCR" onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/understanding-real-time-pcr-tutorial.html' );" href="javascript:void(0);"> <img style="border:none" src="/webroot/web/images/lsr/products/amplification_pcr/category_overlay_icons/global/understanding-real-rime-pcr-trainig-module-thumb.jpg" alt="Understanding Real-Time PCR" /></a></div> <div class="videoDesc"><a title="Understanding Real-Time PCR " onclick="javascript:openAjaxOverlay('/webroot/web/html/lsr/products/amplification_pcr/category_overlay/global/understanding-real-time-pcr-tutorial.html' );" href="javascript:void(0);">Understanding Real-Time PCR</a><br />This video explains the principles behind the polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR), a technique using fluorescent detection of PCR products in real time to track exponential DNA amplification by PCR coupled with analytic methods to calculate the initial number of copies of a target DNA sequence with high sensitivity and accuracy.</div> <div class="clear">&nbsp;</div> </div> Why-is-my-supermix-p_1384537055 Which-real-time-PCR-_1384536897 Why-were-Bio-Rad-rea_1384537662 Are-there-any-perfor_1384537986 Will-my-data-remain-_1384537284 Will-I-see-a-differe_1384540949 Do-levels-of-RFUs-fo_1384541547 What-components-are-_1384541752 What-is-the-Sso7d-fu_1384542041 How-will-Sso7d-fusio_1384542457 Is-there-a-list-of-e_1384542839 Karen Moss Normalization of Real-Time PCR Fluorescence Data with ROX Passive Reference Dye 11/13/13 05:17 PM 11/13/23 05:32 PM 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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