CINXE.COM

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container"><div class="region region-content"><div id="block-system-main" class="block block-system clearfix"><div class="content"><div id="node-20208" class="node node-product-category node-promoted" about="/control-slides-and-control-probes-rnascope" typeof="sioc:Item foaf:Document"><div class="nav-tab-container"><ul class="nav nav-tabs page-tabs" id="myTab"><li class="active"><a href="/control-slides-and-control-probes-rnascope" class="active">Overview</a></a></li><li><a href="/control-slides-0">Control Slides</a></li><li><a href="/control-probes-manual-brown-red">Control Probes: Manual Brown & Red</a></li><li><a href="/control-probes-manual-duplex">Control Probes: Manual Duplex</a></li><li><a href="/control-probes-manual-multiplex">Control Probes: Manual Multiplex</a></li><li><a href="/control-probes-automated-assays-ls">Control Probes: Automated Assays LS</a></li><li><a href="/control-probes-automated-assays-vs">Control Probes: Automated Assays VS</a></li><li><a href="/rnascope%E2%84%A2-pretreatment">RNAscope™ Pretreatment</a></li><li><a href="/PretreatmentOptions">Pretreatment Options</a></li></ul></div><div class="tab-content"><div id="home" class="tab-pane fade in active flexible"><div class="home_content"><div class="flexible_content"><p class="four_flexible">Control slides and control probes to assess technique, sample quality and RNA quality</p><p>Success with any assay begins with good and consistent quality control practices. RNAscope™ <em>in situ</em> hybridization is a nucleic acids-based method in which rigorous controls can be easily incorporated into every assay. ACD recommends two levels of quality control practice:</p><p><strong>Technical assay control check: </strong>The RNAscope™ Assay is an easy single-day assay, similar in workflow to the immunohistochemistry (IHC) workflow. The highly specific and sensitive nucleic acid hybridization is made possible with RNAscope™ technology, which requires close attention to the protocol. To ensure first-time success with specific detection of your intended target using the RNAscope™ assay, ACD strongly encourages all users to perform a technical assay control to ensure that the assay is being performed appropriately. This technical control is easily performed using a cell pellet control sample, tested on two slides with a low-copy housekeeping gene positive control probe and a non-specific bacterial negative control probe. When the assay is run properly, this technical control will show strong positive control probe staining and clean negative control probe staining. We do this with every assay to verify that it is being run with the correct technique.</p><p><strong>Sample/RNA quality control check:</strong> The RNAscope™ Assay has universal assay conditions. Every target-specific probe uses identical hybridization conditions. However, tissue RNA quality and fixation conditions often vary. As a result, it is occasionally necessary to adjust the pretreatment conditions for optimal RNA exposure and detection by RNAscope™ ISH. Empirically, we have observed that the vast majority of properly fixed tissues are suitable for RNA detection with RNAscope™ ISH. ACD provides guidelines on pretreatment conditions for certain tissue types. However, we encourage you to optimize the specific pretreatment conditions for your tissue samples before performing your experiments. Do this by first using the positive and negative control probes on your tissues to verify that you have high positive control signal and no negative control background. If signal is low or there is any background, this is most often improved with adjustment to pretreatment times. Once you have established that sample and RNA quality are good, you can be sure that the RNAscope™ ISH experiments with target-specific probes will be successful.</p></div></div> <script type="text/javascript"> jQuery(document).ready(function($) { $('.nomen-content').hide(); $('.nomen-tabs li').hover(function() { $('.nomen-content').show(); $(this).addClass('active'); $('.nomen-content > div').removeClass('active'); $('.nomen-content #'+$(this).attr('key')).addClass('active'); }, function() { $('.nomen-content').hide(); $('.nomen-tabs li').removeClass('active'); }); $('.nomen-content').hover(function() { $(this).show(); $('li[key='+$(this).children('div.active').attr('id')+']').addClass('active'); }, function() { $(this).hide(); $('.nomen-tabs li').removeClass('active'); }); }); </script> <div class="profile"><div class="additional-detail profile-bottom"><h2>RNAscope Negative Control Probes</h2><p>ACD negative control probes ensure that there is no background staining related to your RNAscope™ assay and that your tissue specimen is appropriately prepared. ACD developed a universal negative control with probes targeting the DapB gene (accession # EF191515) from the <em>Bacillus subtilis</em> strain SMY, a soil bacterium.</p><p>Alternative negative control probes can be made-to-order at your request for your gene of choice in the sense direction (RNAscope probes are antisense) or scrambled probes. We discourage the use of sense probes because occasionally transcription does occur on the opposite strand (and many of these events are not very well documented), in which case the results with what is intended to be a negative control to an antisense RNA may be ambiguous. Alternatively, you can apply probes from unrelated species, for instance use a zebrafish probe on human tissue.</p></div></div><div class="home_content_bottom"><p class="which_rna">Which RNAscope™ Positive Control Probe is right for me?</p><p class="sub_description"></p><div class="classification"><div id="tablefield-wrapper-node-20208-field-table-0" class="tablefield-wrapper"><table id="tablefield-node-20208-field_table-0" class="tablefield tablefield-columns-4 table sticky-enabled"><caption></caption><thead><tr><th class="row_0 col_0" scope="col"></th><th class="row_0 col_1" scope="col"><p>Positive Control Probe Gene</p></th><th class="row_0 col_2" scope="col"><p>Expression Level (copies per cell)*</p></th><th class="row_0 col_3" scope="col"><p>Recommendations</p></th></tr></thead><tbody><tr class="odd"><td class="row_1 col_0"><p>Use with high expression targets</p></td><td class="row_1 col_1"><p>UBC (ubiquitin C)</p></td><td class="row_1 col_2"><p>Medium / High (>20)</p></td><td class="row_1 col_3"><p>UBC is expressed at a medium high level and should be paired with high expressing targets. Applying UBC along side with a low expressing target is not recommended as it can lead to giving false negative results. Because the RNAscope assay is so sensitive, it may be possible to detect UBC RNA even with substantial degradation and under non-optimal assay conditions.</p></td></tr><tr class="even"><td class="row_2 col_0"><p>Most flexible option</p></td><td class="row_2 col_1"><p>PPIB (Cyclophilin B)</p></td><td class="row_2 col_2"><p>Medium (10-30)</p></td><td class="row_2 col_3"><p>PPIB is our recommendation for use as a positive control for most tissues. It is expressed at a sufficiently low level so as to provide a rigorous control for sample quality and technical performance. In the vast majority of studies, if PPIB is positive, then any target probe will detect your target RNA. PPIB is also often used as a reference in RT-PCR.</p></td></tr><tr class="odd"><td class="row_3 col_0"><p>Use with low expression targets</p></td><td class="row_3 col_1"><p>Polr2A (DNA-directed RNA polymerase II subunit RPB1)</p></td><td class="row_3 col_2"><p>Low (3-15)</p></td><td class="row_3 col_3"><p>POLR2A is an additional low copy, rigorous positive control; for very low expressing targets. 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//(Defaults to Chat with an Expert) embedded_svc.settings.disabledMinimizedText = 'Leave a message'; //(Defaults to Agent Offline) embedded_svc.settings.autoOpenPostChat = true; //embedded_svc.settings.loadingText = ''; //(Defaults to Loading) //embedded_svc.settings.storageDomain = 'yourdomain.com'; //(Sets the domain for your deployment so that visitors can navigate subdomains during a chat session) // Settings for Chat embedded_svc.settings.directToButtonRouting = function (prechatFormData) { if (prechatFormData[4].value === "Technical Service") return "5733t000000XZAM"; if (prechatFormData[4].value === "Customer Care") return "5733t000000XZAH"; }; embedded_svc.settings.prepopulatedPrechatFields = { OmniURL__c: window.location.href, ChooseaDepartment__c: 'Technical Service' }; //Sets the auto-population of pre-chat form fields embedded_svc.settings.extraPrechatFormDetails = [{ "label":"Source Domain", "value":window.location.href, "transcriptFields":[ "Source_Domain__c" ], "displayToAgent":true }]; //embedded_svc.settings.fallbackRouting = []; //An array of button IDs, user IDs, or userId_buttonId embedded_svc.settings.offlineSupportMinimizedText = 'Leave a message'; //(Defaults to Contact Us) embedded_svc.settings.enabledFeatures = ['LiveAgent']; embedded_svc.settings.entryFeature = 'LiveAgent'; embedded_svc.init( 'https://biotechne.my.salesforce.com', 'https://biotechne.my.salesforce-sites.com/LiveAgent', gslbBaseURL, '00D500000006gkl', 'ACD_Chat_Automated', { baseLiveAgentContentURL: 'https://c.la1-c1-ia5.salesforceliveagent.com/content', deploymentId: '572380000004CqL', buttonId: '5733t000000cVk9', baseLiveAgentURL: 'https://d.la1-c1-ia5.salesforceliveagent.com/chat', eswLiveAgentDevName: 'EmbeddedServiceLiveAgent_Parent04I3t000000Y7zbEAC_175bb741b99', isOfflineSupportEnabled: true } ); }; if (!window.embedded_svc) { var s = document.createElement('script'); s.setAttribute('src', 'https://biotechne.my.salesforce.com/embeddedservice/5.0/esw.min.js'); s.onload = function() { initESW(null); }; document.body.appendChild(s); } else { initESW('https://service.force.com'); } </script> <script src="//rum-static.pingdom.net/pa-5c133f6e1872b50016000024.js" async></script> <script type="text/javascript">window.NREUM||(NREUM={});NREUM.info={"beacon":"bam.nr-data.net","licenseKey":"NRJS-dd6144c4de227d09eb4","applicationID":"1022125825","transactionName":"YFxbZkpUXBYEVkYNWVkWbEBRGlsLAVBKSkZfSQ==","queueTime":0,"applicationTime":293,"atts":"TBtYEAJOTxg=","errorBeacon":"bam.nr-data.net","agent":""}</script></body> </html>