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Rnascope® Manual Reagents Gene Expression Analysis by RNA in Situ Hybridization

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Rnascope® Manual Reagents Gene Expression Analysis by RNA in Situ Hybridization RNAscope® Manual Reagents Gene Expression Analysis by RNA In Situ Hybridization Get quantitative molecular detection with morphological context in a single assay The RNAscope® assay is the most advanced RNA in situ hybridization (ISH) methodology based on ACD’s patented technology, with signal EQTPMƤGEXMSRERHWMQYPXERISYWFEGOKVSYRHRSMWIWYTTVIWWMSRXLEX advances RNA analysis in tissues and cells. Unique to this technology, the RNAscope®EWWE]HIPMZIVWUYERXMXEXMZIWIRWMXMZIERHWTIGMƤGQSPIGYPEV detection of RNA species on a cell-by-cell basis with morphological context 2018 >650 publications in a single assay. This enables researchers to visualize which genes are expressed, localize where they are expressed, and quantify the level of expression. 2017 Research Areas >450 publications With more than 1,800 publications since 2011, researchers have embraced the RNAscope® technology in a multitude of research areas, showing the universality of the RNAscope® technology. With its unique features and unequal performances, the RNAscope® technology is an essential tool in any research 2016 and pre-clinical research laboratory. >300 publications • Cancer research 2015 • Stem cell >200 publications • Neuroscience • Immunotherapy 2014 • Xenograft models 95 publications • Infectious disease • Preclinical safety assessment 2013 • Gene and cell therapy 47 publications • And more... 2012 11 publications 2011 Advanced Cell Diagnostics 4 publications Publication Count 1,800+* *In November 2018 2 Advanced Cell Diagnostics Explore the RNAscope® World Principle and features of the RNAscope® Technology ...................................................4 A solution for common research challenges ...................................................................6 >650 publications RNAscope® in situ1ERYEP%WWE];SVOƥS[ .....................................................................8 Step 01. Permeabilize Step 04. Visualize 8MWWYIWIGXMSRWSVGIPPWEVIƤ\IHSRXSWPMHIWERH Each punctate dot signal represents a single pretreated with RNAscope® Pretreatment Kit to target RNA molecule and can be visualized YRQEWOXEVKIX62%ERHTIVQIEFMPM^IGIPPW with a standard microscopes. Pretreatment Reagents / pg. 8 pg. 14 Step 02. Hybridize Step 05. Quantify Double Z probe pools are hybridized 7MRKPIQSPIGYPIWMKREPWEVIUYERXMƤIHSRE to target RNA molecules. cell-by-cell basis by manual counting or automated image analysis with RNAscope® and HALOTM and other RNAscope® Target Probes / pg. 9 software. RNAScope® Control Probes / pg. 9 RNAscope® & HALOTM software/ pg. 14 Step 03. Amplify 7IUYIRXMEPL]FVMHM^EXMSRSJEQTPMƤIVW and labeled probe(s). RNAscope® Detection Reagents / pg. 10 Accessories / pg. 13 RNAscope® Manual Solutions 3 Principle and features of RNAscope® Technology Innovative solution for single RNA molecule detection ERHUYERXMƤGEXMSRMRWMRKPIGIPPW RNAscope® probe design RNAscope® probe hybridization and E Q T P M Ƥ G E X M S R SGGYVWEWEGEWGEHISJIZIRXW A standard target probe consists of a pool of 20 double Z Step 1: Hybridization of 20 ZZ probe pairs to the RNA target probes targeting a region of 1,000 bases. Each Z target probe Step 2:,]FVMHM^EXMSRSJXLITVIEQTPMƤIVXSXLIYTTIV contains three elements: The lower region is complementary regions of the Z probe pairs XSXLIXEVKIX62%ERHMWWIPIGXIHJSVXEVKIXWTIGMƤGL]FVMHM^E- tion and uniform hybridization properties. A spacer sequence Step 3: ,]FVMHM^EXMSRSJQYPXMTPIEQTPMƤIVWTIVTVIEQTPMƤIV PMROWXLIPS[IVVIKMSRXSERYTTIVVIKMSR8LIX[SEHNEGIRX Step 4: ,]FVMHM^EXMSRSJQYPXMTPIPEFIPIHTVSFIWTIVEQTPMƤIV upper regions from a double Z target probe forms a 28 base Serial hybridization events - 20 ZZ probe pairs, multiple FMRHMRKWMXIJSVXLITVIEQTPMƤIV EQTPMƤIVWQYPXMTPIPEFIPIHTVSFIWVIWYPXMRL]FVMHM^EXMSRSJ Two independent Z probes, designed as probe pairs, need to thousands of labeled probes per RNA target. hybridize to the target sequence in tandem in order to enable FMRHMRKSJXLITVIEQTPMƤIV %WMRKPI>TVSFIL]FVMHM^EXMSRSRXSERSRWTIGMƤG62%XEVKIX can happen, but the resulting hybridization of the pre-ampli- ƤIVSRXSXLIYTTIVVIKMSRSJEWMRKPI>[MPPFIYRWXEFPIERH therefore will be removed during the wash steps. This design IRWYVIWEPS[FEGOKVSYRHRSMWIPIZIP #ORNKƒGT Probe Design 2TGCORNKƒGTDKPFKPI site,14 bases Linker Target binding site 18-25 bases Z target probe design Complementary bases Hybridization requires 2TGCORNKƒGT double Z binding Complementary bases #ORNKƒECVKQP Hybridization RNA target 4 Advanced Cell Diagnostics RNAscope® signal detection &IRIƤXWSJXLI62%WGSTI® technology ŵ,MKLWIRWMXMZMX]8LIWIVMEPWMKREPEQTPMƤGEXMSR design increases sensitivity such that a single Labeled probes contain either a chromogenic enzyme or RNA molecule can be detected. EƥYSVSTLSVIWMKREPKIRIVEXMRKSRITYRGXEXIHSXTIV62% ŵ,MKLWTIGMƤGMX]Proprietary probe design target. (See page 14 - Visualize Step). IRWYVIWXEVKIXWTIGMƤGFMRHMRK[LMPIXLIHSYFPI ,]FVMHM^EXMSRSJSRP]XLVII>TVSFITEMVWMWWYJƤGMIRXXSSFXEMR >TVSFIHIWMKRTVIZIRXWWMKREPEQTPMƤGEXMSRSJ a detectable signal by a standard microscope. RSRWTIGMƤGL]FVMHM^EXMSR • Morphological context: Spatial resolution of gene expression in the complex tissue environmet creates a spatial map • Per-cell quantitation: High sensitivity combined with morphological context results in single-molecule detection at single-cell resolution. ŵ9RMZIVWEP;SVOWJSVZMVXYEPP]%2=KIRIJVSQ %2=WTIGMIWMR%2=XMWWYI #ORNKƒGT Labeled probe binding site Labeled probe 2TGCORNKƒGTDKPFKPI 2TGCORNKƒGT #ORNKƒECVKQP RNAscope® Manual Solutions 5 A solution for common research challenges 6ETMHZEPMHEXMSRSJFMSQEVOIVHMWGSZIV] RNA expression analysis to complement ;LIXLIV]SYEVIGLEVEGXIVM^MRKFMSQEVOIVWHMWGSZIVIH or combine with IHC-based protein by single-cell RNA sequencing, NGS, microarray or high analysis throughput qPCR, the RNAscope® XIGLRSPSK]MWEUYMGOERH )\EQMREXMSRSJTVSXIMREWEFMSQEVOIV[MXL IEW]XSSPXSYWIEGVSWWXLIHMJJIVIRXWXEKIWSJXLIFMSQEVOIV immunohistochemistry (IHC) technique is a widely used ® validation. With the RNAscope technology you have access and accepted approach for diagnosis, prognosis, and to unique gene expression information with morphological therapy development for clinical diseases. However, the context: digital RNA expression at single cell level in complex number of high quality and reliable antibodies is limited tissue structure Theses. and IHC is not without issues, and the use of sometimes TSSVP]GLEVEGXIVM^IHERXMFSHMIWERHMRWYJƤGMIRXSZIVEPP standardization often leads to questionable results. At the contrary, RNAscope® technology is based on probe HIWMKRIHXSFILMKLP]WTIGMƤGXSXLIXEVKIXERHVITVSHYGMFP] manufactured. With a unique and reproducible protocol, RNAscope® assay is an ideal solution to validate, supplement or combine with IHC. FIGURE 1. RNAscope®EWWE]EWMQTPIƤVWXƤPXIVMRXEVKIXZEPMHEXMSR 6 Advanced Cell Diagnostics Non-coding RNA expression -RXLIQSWXVIGIRXWXEXMWXMGWJVSQXLI+)2'3()TVSNIGX 8LIHMWGSZIV]SJETVIZMSYWP]YRORS[RYRMZIVWISJPRG62%W (v29, October 2018), the human genome contains 23,643 has created an unprecedented demand for effective RNA in non-coding RNA (ncRNA) genes, surpassing the number situL]FVMHM^EXMSRXSSPW9RPMOITVSXIMRGSHMRKKIRIWJSV[LMGL of protein-coding genes (19,940). Of the non-coding RNA immunohistochemistry (IHC) and RNA in situ hybridization are species, some 30% (7,577) are less than 200 bases long, GSQTPIQIRXEV]JSVQETTMRKKIRII\TVIWWMSRXSWTIGMƤGGIPPW termed as small non-coding RNA. They comprise of transfer in situ, lncRNA expression can only be investigated by RNA RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs in situ hybridization. The generally lower expression levels of such as snoRNAs, microRNAs, siRNAs, snRNAs, exRNAs lncRNAs than their protein coding counterparts demand the and piRNA. About 60% (16,066) of the non-coding RNAs are highest sensitivity from RNA in situ hybridization methods. longer than 200 bases and are operationally designated as The single-molecule sensitivity and rapid assay development long non-coding RNAs (lncRNAs).The functions of lncRNAs XMQI [IIOW SJ%'(ŭW62%WGSTI®XIGLRSPSK]QEOI are still characterized; their abundance and diversity add to RNAscope® ideally suited for localizing lncRNAs expression to the challenge. Some lncRNAs have been shown to regulate WTIGMƤGGIPPX]TIWERHWYFGIPPYPEVWXVYGXYVIW62%WGSTI® in gene expression through a diversity of mechanisms and situ assays will undoubtedly accelerate lncRNA research and TPE]MQTSVXERXVSPIWMRGLVSQEXMRQSHMƤGEXMSR ,38%-6 become an indispensable tool for lncRNA-based molecular transcriptional and post-transcriptional regulation (ZEB2). diagnostics. Dysregulation of lncRNA is being found to have relevance not only in tumorigenesis, but also to neurological, cardiovascular, developmental and other diseases. “ This technology allows us to directly visualize gene expression in the target 1A3 tissue of interest – for example, within the same sample we can tell whether gene overexpression occurs in benign prostate glands, high grade prostatic intraepithelial neoplasia (HGPIN – a pre-cancerous state) or prostate cancer.” Dr. Mehra, Clinical Assistant Professor of Pathology at Michigan Center for Translational Pathology Hs-PD1 Hs -TIM3 Hs-PDL1 CD45 (Ab) *-+96)7MRKPIGIPPGSI\TVIWWMSRTVSƤPIWSJGLIGOTSMRXQSPIGYPIWMR FIGURE 3. Non-coding PCA3 transcript detection in whole prostate tumor selected non-small cell lung cancer (NSCLC) cores. whole tissue section using RNAscope® technology. RNAscope® Manual Solutions 7 RNAscope® in situ Manual Assay ;SVOƥS[ERHEWWSGMEXIHTVSHYGXW Step 01. RNAscope® Pretreatment Reagents Permeabilize Optimized permeabilization for optimal target accessibility.
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