CINXE.COM

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<img class="img-banner" src="https://acdbio.com/sites/default/files/science-banner.jpg"><h1 class="general-title"> NGS Validation, RNA-Seq, NanoString Single-Cell RNA-seq, Transcriptomics, Validation of RNA Sequencing</h1><h2 class="banner-sub-title-seo">RNAscope™ Applications</h2></div><div class="content-container container newapplication"><div id="node-112528" class="node node-application" about="/science/applications/research-solutions/ngsht-transcriptomic-validation-rna-seq" typeof="sioc:Item foaf:Document"><div class="nav-tab-container"><ul class="nav nav-tabs page-tabs application-bxslider" id="myTab"><li><a href="/science/applications/research-solutions/long-non-coding-rna">Long non-coding RNA</a></li><li><a href="/science/applications/research-solutions/single-cell-analysis">Single Cell Analysis</a></li><li><a href="/science/applications/research-solutions/gene-fusions">Gene Fusions</a></li><li class="active"><a href="/science/applications/research-solutions/ngsht-transcriptomic-validation-rna-seq" class="active">NGS/HT Transcriptomic Validation (RNA-Seq)</a></li><li><a href="/science/applications/research-solutions/circular-rna">Circular RNA</a></li><li><a href="/science/applications/research-solutions/anatomic-pathology-panels">Anatomic Pathology Panels</a></li><li><a href="/science/applications/research-solutions/mirna">miRNA</a></li></ul></div><div class="tab-content"><div id="tab0" class="tab-pane fade in active profile"><h1 class="top-profile">RNAscope™ ISH as a validation tool for high-throughput transcriptomic analyses</h1><div class="sample-image"></div><div class="profile-content application_node_112528"><div class="field field-name-body field-type-text-with-summary field-label-hidden"><div class="field-items"><div class="field-item even" property="content:encoded"><div class="profile-bottom"><p>High-throughput transcriptomic analyses, such as microarray, RNA sequencing (RNA-Seq) and NanoString nCounter, enable researchers to study the complete set of RNA transcripts produced by the genome. These analyses can generate a wealth of data, but most often need to be validated within the tissue microenvironment. RNAscope™ ISH and the recently introduced (Aug 2016) BaseScope™ ISH assays can both be applied as methods to validate high-throughput findings at the single cell level with spatial information in tissue.</p><ul><li><strong>Confirmation of results</strong> - RNAscope assays have been used in numerous publications to confirm the results of high-throughput analyses and provide further information on the cellular localization of RNA species</li><li><strong>Alternative splicing</strong> - BaseScope ISH is capable of detecting splice variants using probes that span the exon junctions present in a variant. Information derived from RNA-seq on alternative splicing events can be validated in cells and tissues by BaseScope ISH.</li><li><strong>Co-expression - </strong>RNAscope Multiplex Fluorescent Assays and Duplex Chromogenic Assays are routinely applied to simultaneously visualize expression of up to three RNA targets. Co-localization of two or three specific markers in the same or different cells can be determined by RNAscope ISH.</li></ul></div><div class="app-grey-bg"><div class="app-content-bg"><p><strong>Validation of Single-Cell RNA-seq</strong> - Silberstein <em>et al.</em> applied this technology to single cells to identify the secreted factors produced by transplanted niche cells that regulate stem cell function. RNAscope ISH was used to validate the expression of IL18 proximal to the transplantation site.</p><p><strong><em>Publication:</em></strong></p><p><a href="http://www.ncbi.nlm.nih.gov/pubmed/27524439"><em>Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators</em></a> Silberstein et al. <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=27524439" title="Cell stem cell.">Cell Stem Cell.</a> 2016 Aug 9. pii: S1934-5909(16)30199-0. doi: 10.1016/j.stem.2016.07.004. PMID:<a href="http://www.ncbi.nlm.nih.gov/pubmed/27524439">27524439</a></p><h5 style="border: currentColor; border-image: none; line-height: 1.1; font-family: Roboto, sans-serif; font-size: 24px; font-weight: lighter; margin-top: 20px; margin-bottom: 20px; box-sizing: border-box;"><strong>Researchers in the Spotlight--Download interviews to read how researchers use RNAscope ISH to validate RNA-seq results.</strong></h5><p style="margin: 0px 0px 20px; padding: 0px; line-height: 30px; font-family: Roboto, sans-serif; font-size: 18px; box-sizing: border-box;"><span style="font-size: 16px;"><span style="padding: 10px 0px 0px; font-weight: 700; display: inline-block; box-sizing: border-box;">Interview with Dr Rohit Mehra:</span><br />Realising the potential of Iong noncoding RNA as a cancer biomarker - From NGS discovery to validation with RNA <em>in situ</em> Hybridization<br /><u class="download-pdf-app"><a class="down-pdf-btn" href="https://acdbio.com/system/files_force/gated/ACD__Researcher_in_spotlight_Rohit_Mehra_50_006.pdf?download=1">Download Interview </a> </u> </span></p><p style="margin: 0px 0px 20px; padding: 0px; line-height: 30px; font-family: Roboto, sans-serif; font-size: 18px; box-sizing: border-box;"><span style="font-size: 16px;"><span style="padding: 10px 0px 0px; font-weight: 700; display: inline-block; box-sizing: border-box;">Interview with Dr Nallasivam Palanisamy:</span><br />Development of Prostate Cancer Personalized Medicine - Advancing analysis of prostate tumor molecular heterogeneity by combined immunohistochemistry and novel RNA <em>in situ</em> hybridization<br /><u class="download-pdf-app"><a class="down-pdf-btn" href="https://acdbio.com/system/files_force/gated/ACD_Researcher_Spotlight__Nalla_Palanisamy_50_005.pdf?download=1">Download Interview </a> </u> </span></p><p style="margin: 0px 0px 20px; padding: 0px; line-height: 30px; font-family: Roboto, sans-serif; font-size: 18px; box-sizing: border-box;"><span style="font-size: 16px;"><span style="padding: 10px 0px 0px; font-family: Roboto, sans-serif; font-weight: 700; display: inline-block; box-sizing: border-box;">Interview with Dr Alexy Pronin:</span><br />Exploring GPCR expression in the eye. From discovery with NGS to spatial and quantitatvie expression confirmation with RNAscope technology<br /><u class="download-pdf-app"><a class="down-pdf-btn" href="https://acdbio.com/system/files_force/gated/ACD_Researcher_Spotlight__Alexey_Pronin_50_002.pdf?download=1">Download Interview </a> </u> </span></p></div></div><div class="app-white-bg"><div class="app-content-bg"><p><strong>Validation of NanoString nCounter –</strong> Chen <em>et al. </em>performed a NanoString assay to identify genes differentially expressed between control and LKB1 mutant lung cancer samples. The RNAscope ISH assay was used to validate these results, showing that the lncRNA LINC00473 is associated with LKB1 inactivation in NSCLC. Given the poor specificity of LKB1 antibodies, this paper demonstrates that <em>LINC00473</em> could be used as a surrogate biomarker for LKB1 in lung cancer samples.</p><p><strong><em>Publication:</em></strong></p><p><a href="http://www.ncbi.nlm.nih.gov/pubmed/27140397"><em>cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth</em></a> Chen <em>et al.</em> <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=27140397" title="The Journal of clinical investigation.">J Clin Invest.</a> 2016 Jun 1;126(6):2267-79. doi: 10.1172/JCI85250 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/27140397">27140397</a></p></div></div><div class="app-grey-bg"><div class="app-content-bg"><p><strong>Validation of lncRNA Microarray</strong> – In a recent study, lncRNA microarrays identified over 2800 lncRNAs that were differentially expressed between triple-negative breast cancer (TNBC) tissue and normal adjacent tissue. Lin <em>et al. </em>used RNAscope ISH to confirm that expression of the lncRNA LINK-A is indeed significantly increased in TNBC tissues compared to adjacent normal tissues, and also were able to further localize the expression to the cytoplasm and close to the cellular membrane.</p><p><strong>Publication:</strong> </p><p><a href="http://www.ncbi.nlm.nih.gov/pubmed/26751287"><em>The </em><em>LINK-A </em><em>lncRNA activates normoxic HIF1</em><em>α</em><em> signalling in triple-negative breast cancer</em></a> Lin <em>et al.</em> <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=26751287" title="Nature cell biology.">Nat Cell Biol.</a> 2016 Feb;18(2):213-24. doi: 10.1038/ncb3295 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/26751287">26751287</a></p></div></div><div class="app-white-bg"><div class="app-content-bg"><p><strong>Validation of Digital Transcriptome Subtraction (DTS) —</strong> This recently developed method has been used to identify pathogenic viruses in cancer, but can also be applied to non-cancer related diseases. Whole transcriptome sequencing is applied to a tumor sample followed by in silico removal of host sequence fragments. The remaining sequences are aligned and blasted against known pathogen sequences to identify candidate sequences present in the sample. Highly sensitive methods such as RNAscope ISH are then performed to validate the presence of the pathogenic sequence. Cimino<em> et al</em>. described this application in their 2014 publication. </p><p><strong>Publication:</strong> </p><p><a href="http://www.ncbi.nlm.nih.gov/pubmed/24704430"><em>Detection of viral pathogens in high grade gliomas from unmapped next-generation sequencing data</em></a><em> Cimino et al. </em><a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=24704430" title="Experimental and molecular pathology.">Exp Mol Pathol.</a> 2014 Jun;96(3):310-5. doi: 10.1016/j.yexmp.2014.03.010 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24704430">24704430</a></p></div></div></div></div></div><div class="view view-application view-id-application view-display-id-block_8 view-dom-id-56aa988c571aaf6a2e060f8ef0dc96bf"></div><div class="view view-application view-id-application view-display-id-block_9 view-dom-id-c68672d43265a2bb2072eff3a5d3dcd4"><div class="view-content"><style type="text/css"> .view-id-application.view-display-id-block_9 .row.immuno-watch { margin-top: 15px; margin-bottom: 15px; } </style></div></div></div><div class="sample-image"><div class="profile-bottom"></div></div><div class="spotlight"><div class="profile-bottom publication"><p>RNAscope ISH has been cited in many publications as a validation tool applied downstream of discoveries made with NGS, Microarrays, NanoString nCounter.</p><div class="download-pdf"> <a href="https://acdbio.com/science/scientific-resources/publications/single%20cell"> Read Abstracts </a></div></div></div><div class="call-to-action"><div class="profile-bottom"><h2>Validate and localize expression of your transcriptomic results. 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background-color: inherit; border-style: none; color: #fff; margin: 4px 20px; font-size: large; } .embeddedServiceLiveAgentStateChatHeader.alert .message { margin: 0; } .embeddedServiceLiveAgentStateChatHeader:not(.alert) .message { margin: 0; width: auto; } span#helpButtonSpan { padding: 0 50px 0 30px; } .embeddedServiceLiveAgentStateChatHeaderOption .optionName { color: #ffffff; } /* hide the URL capture field and label */ #OmniURL__c { display: none; } .prechatUI .fieldList li:nth-child(6) label { display: none; } /* Added 10px margin-top to center header text */ .headerText { margin-top: 10px; } </style>  <style type='text/css'> #snapins_invite { position: fixed !important; background-color: #FFFFFF; font-family: "Arial", sans-serif; overflow: visible; border-radius: 8px; visibility: hidden; } .embeddedServiceInvitation { background-color: transparent; max-width: 290px; max-height: 210px; -webkit-box-shadow: 0 7px 12px rgba(0, 0, 0, 0.28); -moz-box-shadow: 0 7px 12px rgba(0, 0, 0, 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max-height: 210px; } } .embeddedServiceInvitation > .embeddedServiceInvitationHeader { width: inherit; height: 32px; line-height: 32px; padding: 10px; color: #FFFFFF; background-color: #222222; overflow: initial; display: flex; justify-content: space-between; align-items: stretch; border-top-left-radius: 8px; border-top-right-radius: 8px; } .embeddedServiceInvitationHeader #embeddedServiceAvatar { width: 32px; height: 32px; border-radius: 50%; } .embeddedServiceInvitationHeader .embeddedServiceTitleText { font-size: 18px; color: #FFFFFF; overflow: hidden; word-wrap: normal; white-space: nowrap; text-overflow: ellipsis; align-self: stretch; flex-grow: 1; max-width: 100%; margin: 0 12px; } .embeddedServiceInvitationHeader .embeddedServiceCloseIcon { border: none; border-radius: 3px; cursor: pointer; position: relative; bottom: 3%; background-color: transparent; width: 32px; height: 32px; font-size: 23px; color: #FFFFFF; } .embeddedServiceInvitationHeader .embeddedServiceCloseIcon:focus { outline: none; } .embeddedServiceInvitationHeader .embeddedServiceCloseIcon:focus::before { content: " "; position: absolute; top: 11%; left: 7%; width: 85%; height: 85%; background-color: rgba(255, 255, 255, 0.2); border-radius: 4px; pointer-events: none; } .embeddedServiceInvitationHeader .embeddedServiceCloseIcon:active, .embeddedServiceCloseIcon:hover { background-color: #FFFFFF; color: rgba(0,0,0,0.7); opacity: 0.7; } .embeddedServiceInvitation > .embeddedServiceInvitationBody { background-color: #FFFFFF; max-height: 110px; min-width: 260px; margin: 0 8px; font-size: 14px; line-height: 20px; overflow: auto; } .embeddedServiceInvitationBody p { color: #333333; padding: 8px; margin: 12px 0; } .embeddedServiceInvitation > .embeddedServiceInvitationFooter { width: inherit; color: #FFFFFF; text-align: right; background-color: #FFFFFF; padding: 10px; max-height: 50px; border-bottom-left-radius: 8px; border-bottom-right-radius: 8px; } .embeddedServiceInvitationFooter > 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