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:,]FVMHMÊXMSRSJXLITVIEQTPMƤIVXSXLIYTTIV contains three elements: The lower region is complementary regions of the Z probe pairs XSXLIXEVKIX62%ERHMWWIPIGXIHJSVXEVKIXWTIGMƤGL]FVMHMÊ- tion and uniform hybridization properties. A spacer sequence Step 3: ,]FVMHMÊXMSRSJQYPXMTPIEQTPMƤIVWTIVTVIEQTPMƤIV PMROWXLIPS[IVVIKMSRXSERYTTIVVIKMSR8LIX[SEHNEGIRX Step 4: ,]FVMHMÊXMSRSJQYPXMTPIPEFIPIHTVSFIWTIVEQTPMƤIV upper regions from a double Z target probe forms a 28 base Serial hybridization events - 20 ZZ probe pairs, multiple FMRHMRKWMXIJSVXLITVIEQTPMƤIV EQTPMƤIVWQYPXMTPIPEFIPIHTVSFIWVIWYPXMRL]FVMHMÊXMSRSJ 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]FVMHMÊXMSRSRXSERSRWTIGMƤG62%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.

In order to perform the RNAscope® assay, start with properly RNAscope® 2.5 Pretreatment Reagents provide improved prepared and pretreated samples. accessibility to target RNA reducing the time and effort Sample preparation and pretreatment include the in assay optimization. These reagents include hydrogen following steps: TIVS\MHIXSFPSGOIRHSKIRSYWTIVS\MHEWIEGXMZMX]%HHMXMSREP pretreatment reagents such as target retrieval and protease • Fixation of cells or tissues if needed (fresh-frozen, cultured pretreatment reagents allow the RNAscope® probes to better cells, etc.) EGGIWWXLI62%FVIEOMRKGVSWWPMROWXLEXGSYPHSGGYV[MXLXLI ŵ(ITEVEJƤRM^EXMSRMJRIIHIH **4) XMWWYIHYVMRKƤ\EXMSRERHTIVQIEFMPM^MRKGIPPWXSEPPS[XEVKIX accessibility. Pretreatment reagents are available and suitable • Applying pretreatment reagents included in the JSVQYPXMTPIXMWWYIX]TIWMRGPYHMRKJSVQEPMRƤ\IHTEVEJƤR RNAscope® Reagent Kit embedded (FFPE) tissue including archival tissue, fresh frozen ** XMWWYIƤ\IHJVS^IRXMWWYIXMWWYIQMGVSEVVE] 81% ERH cell preparations. RNAscope® 2.5 Universal Pretreatment Reagents is VIGSQQIRHIH[LIR[SVOMRKSVW[MXGLMRKFIX[IIRHMJJIVIRX tissue types such as fresh-frozen or FFPE or cultured cells. It GSRXEMRWEPPXLITVIXVIEXQIRXVIEKIRXWMRSRIOMX

® • RNAscope Hydrogen Peroxide (H202) • RNAscope® Target Retrieval • RNAscope® Protease Plus • RNAscope® Protease III For further information on our pretreatment reagents please visit acdbio.com/pretreatment FIGURE 4. Ppib detection (red punctate dots) in mouse intestine using RNAscope® 2.5 HD Assay-RED.

For more information request or download the RNAscope® Reference Guide containing guidelines and protocols for obtaining optimal RNAscope® in situ hybridization results with any tissue type from any species. acdbio.com/referenceguide

8 Advanced Cell Diagnostics RNAscope® Target Probes Step 02. 9RMUYITVSFIHIWMKRTVSZMHIWLMKLP]WTIGMƤGL]FVMHM^EXMSRXS Hybridize the target molecule.

RNAscope® Catalog Target Probes RNAscope® Made-to-Order Target Probes Using the proprietary ACD RNAscope® Probe Design If ACD catalog probes are not available for your gene of algorithm, we design double-Z oligo probe pools that hybridize MRXIVIWX[IGERGVIEXIRI[TVSFIW[MXLMRX[S[IIOWYWMRK XS]SYVWTIGMƤG62%XEVKIXSJMRXIVIWX;IGERHIWMKRTVSFI public or proprietary sequences. ACD probe design algorithm TSSPWJSVZMVXYEPP]%2=KIRIMR%2=KIRSQIJSVMRXIVVSKEXMSR can also accommodate non-standard designs such as probe MR%2=XMWWYI8LITVSFITSSPWGSRWMWXSJTVSTVMIXEV] TSSPWJSVHIXIGXMSRSJJYWMSRKIRIWWTIGMIWWTIGMƤGHIXIGXMSR SPMKSRYGPISXMHIWHIWMKRIHJSVHIXIGXMRKWTIGMƤGXEVKIXW TEKI SJFMSQEVOIVWMR\IRSKVEJXWSVER]SXLIVRSRWXERHEVH 4-5). Every target probe pool also contains a tag that enables application in any species. Standard and non-standard XLIEWWSGMEXIHXEVKIXXSFIZMWYEPM^IHMREWTIGMƤGŰGSPSV RNA ISH probe pools can be designed for use with any of channel” under the microscope (page 12). our RNAscope® Reagent Kits, including singleplex, duplex, Select from our growing catalog of over 20,000 in situ QYPXMTPI\QERYEPSVEYXSQEXIHEWWE]GSRƤKYVEXMSRW hybridization target probe pools for mRNA and long Interested in custom probes? Tell us your gene of interest and noncoding RNA (lncRNA). Our RNA ISH probe pools span let’s get started: acdbio.com/target-probes-made-order a variety of species including human, mouse, rat, dog, cow, ^IFVEƤWLVEFFMXTMKGLMGOIRQSROI]W,4:,-:,':ERH many others. Search for an assay targeting your gene of interest at acdbio.com/probesearch Submit Sequence Design Manufacturing Shipping & Order 2 Weeks

*-+96)%RIEW]ERHUYMGOHIWMKRERHQERYJEGXYVMRKTVSGIWWJSVLMKLP] WTIGMƤGERHVITVSHYGMFPI62%WGSTI® probes.

Ensure your success with good qualilty controls

RNAscope® Control Probes RNAscope® Control Slides In addition to target probes, we also provide species- The RNAscope® control slides are essential to verify assay WTIGMƤGLSYWIOIITMRKKIRITSWMXMZIGSRXVSPTVSFIWERH GSRHMXMSRW8LIƤVWXVYR[SYPHWIVZIEWEXIGLRMUYIUYEPMX] (ET&RIKEXMZIGSRXVSPTVSFIWHIWMKRIHXS[SVO[MXL GSRXVSPGLIGOERHWLSYPHFIVYR[MXLXLIEWWE]YWMRK RNAscope® Reagent Kits. The positive control probes control probes prior to using your samples and target span from high to very low levels of expression, probes. Two types of control slides are offered: Human providing appropriate experimental controls for RNA control slides contains FFPE cultured cell pellets of human in situL]FVMHM^EXMSRERHIRWYVMRKLMKLGSRƤHIRGI HeLa cells and mouse control slides contains FFPE [LIR[SVOMRK[MXLZEV]MRKSVYRORS[RPIZIPWSJKIRI cultured cell pellets from mouse NIH 3T3 cells. I\TVIWWMSR7IISYVPMWXSJWTIGMIWWTIGMƤGGSRXVSPTVSFIW at acdbio.com/controlprobes

RNAscope® Manual Solutions 9 RNAscope® in situ Manual Assay ;SVOƥS[ERHEWWSGMEXIHTVSHYGXW

Step 03. RNAscope® Reagents Amplify Multiplex your possibilities from single to 4-plex analysis.

RNAscope® 2.5 HD Reagent Kit-BROWN The RNAscope® 2.5 HD Reagent Kit-RED TheMHIEPWXEVXIVOMXJSVƤVWXXMQIYWIVWERHMWYRMZIVWEPMR %*EWX6IHH]I[LMGLSJJIVWELMKLIVGSRXVEWXERHMWXLIƤVWX ETTPMGEXMSRW8LMWZIV]VSFYWXEWWE]KMZIWLMKLHIƤRMXMSR choice for in situ hybridization applications where chromogenic staining results, which can be archived permanently due to the staining with DAB is less desirable, such as staining of highly permanent staining. The chromogen diaminobenzidine (DAB) TMKQIRXIHPYRKPMZIVVIXMREERHWOMRXMWWYIWTIGMQIRW%'( used in the assay is the standard in molecular pathology and also recommends this assay for detection of target genes suitable for a wide range of sample types as well as readily where a lower expression is anticipated, as the red dots stand ZMWMFPIYRHIVEWXERHEVHFVMKLXƤIPHQMGVSWGSTI out more clearly against the hematoxylin staining and are more The RNAscope® 2.5 HD Reagent Kit-BROWN is ideal for VIEHMP]MHIRXMƤEFPIYRHIVEWXERHEVHFVMKLXƤIPHQMGVSWGSTI detection of target genes with anticipated low expression 8LVIIEPXIVREXMZIGSRƤKYVEXMSRWSJXLMWOMXEVIEZEMPEFPIXSIREFPI JYPP]EYXSQEXIH[EPOE[E]-7,WSPYXMSRW PIZIPW ũGSTMIWTIVGIPP 8LVIIEPXIVREXMZIGSRƤKYVEXMSRWSJ XLMWOMXEVIEZEMPEFPIXSIREFPIJYPP]EYXSQEXIH[EPOE[E]-7, • RNAscope® VS Universal AP Reagent Kit-RED for use on the solutions: (-7'3:)6=9086%EYXSQEXIHXMWWYIWXEMRMRKW]WXIQWF] Roche Tissue Diagnostics. • RNAscope® VS Universal HRP Reagent Kit-BROWN for YWISRXLI(-7'3:)6=9086%EYXSQEXIHXMWWYIWXEMRMRK ŵ62%WGSTIo07\6IEKIRX/MX4VIƤPPIH6)(OMXWJSVXLI systems by Roche Tissue Diagnostics. seamless use on the use on the Leica Biosystems’ BOND Rx System. ŵ62%WGSTIo07\6IEKIRX/MX4VIƤPPIH&63;2OMXWJSVXLI seamless use on the use on the Leica Biosystems’ BOND Rx ŵ62%WGSTIo076IEKIRX/MX6)(OMXWJSVXLIWIEQPIWWYWI System. on the use on the Leica Biosystems’ BOND Rx System ŵ62%WGSTIo076IEKIRX/MX&63;2OMXWJSVXLIWIEQPIWW For further information on our automated solutions please visit use on the use on the Leica Biosystems’ BOND Rx System. acdbio.com/automated-assays For further information on our automated solutions please visit acdbio.com/automated-assays

FIGURE 6. ERB2 detection (brown punctate dots) in uman breast cancer FIGURE 7. PDL1 detection (red punctate dots) in lung cancer sample using sample using RNAscope® 2.5 HD Reagent Kit-BROWN. RNAscope® 2.5 HD Reagent Kit-RED.

10 Advanced Cell Diagnostics RNAscope® 2.5 HD Duplex Assay RNAscope® Multiplex Fluorescent Assay Designed for simultaneous in situ detection of two RNA species. The RNAscope® Multiplex Fluorescent assays provide the same Common applications include co-localization studies to map exceptional sensitivity as our singleplex assays, allowing single- co-expression of two targets within the same cellular context molecule detection of up to four RNA targets simultaneously. IKWIGVIXIHPMKERHERHMXWVIGITXSV SVXSTVSƤPIKIRI The RNAscope® Multiplex Fluorescent assays are ideal for I\TVIWWMSRMREWTIGMƤGGIPPX]TII\TVIWWMRKEORS[RQEVOIV co-localization studies of any genes in nearly any tissue-type IKEWTIGMƤGWXIQGIPPQEVOIV 8SHMWXMRKYMWLFIX[IIR YWMRKƥYSVIWGIRXPEFIPW%'(SJJIVWX[SX]TIWSJQYPXMTPI\ the two chromogenic colors, ACD has employed the naming ƥYSVIWGIRXEWWE]W8LI62%WGSTIo*PYSVIWGIRX%WWE]SYV convention of Channel 1 (C1) to refer to green and Channel 2 ƤVWXKIRIVEXMSREWWE]MWEREPPMRSRIOMXMHIEPJSVJVIWLJVS^IR (C2) to Fast Red, hence RNAscope® probe pool names often XMWWYIWEQTPIWERHGYPXYVIHGIPPWEPWSGSQTEXMFPI[MXLƤ\IH include C1 or C2. The stained slides are visualized with bright- frozen samples. The RNAscope® Multiplex Fluorescent Assay ƤIPHQMGVSWGSTIW ZMHIEPJSV**4)ERHƤ\IHJVS^IRWEQTPIWERHGSQTEXMFPI[MXL all sample preparation methods, is a TSA-based assay ideal for **4)XMWWYIWERHVIUYMVIW4IVOMR)PQIVŭWTVSTVMIXEV]8]VEQMHI 8[SEPXIVREXMZIGSRƤKYVEXMSRWSJXLMWOMXEVIEZEMPEFPIXSIREFPI 7MKREP%QTPMƤGEXMSRXIGLRSPSK] 87%Ǝ SV3TEPƎ(]IWJSV JYPP]EYXSQEXIH[EPOE[E]-7,WSPYXMSRW visualization. ŵ62%WGSTIo:7(YTPI\%WWE]JSVYWISRXLI(-7'3:)6= ULTRA automated tissue staining systems by Roche Tissue For a deatiled comparison of those two assays please have a Diagnostics. PSSOEXTEKI • RNAscope® 2.5 LS Duplex Assay for use on the Leica Biosystems’ BOND Rx System. For further information on our Leica automated solutions please visit For further information on our Leica automated solutions EGHFMSGSQVREWGSTIoPWQYPXMTPI\ƥYSVIWGIRXEWWE] please visit acdbio.com/automated-assays

FIGURE 8. Expression analysis of PD-L1 (green) and CD8a (red) in human FIGURE 9. Expression analysis of Drd1 (red) and Drd2 (green) in mouse brain lung cancer tissue using RNAscope® 2.5 HD Duplex Assay. striatum tissue tissue using RNAscope® Multiplex Fluorescent Assay v2

RNAscope® Manual Solutions 11 RNAscope® in situ Manual Assay ;SVOƥS[ERHEWWSGMEXIHTVSHYGXW

Step 03. RNAscope® Reagents Amplify 3RIEWWE]JSVWLSVXWIUYIRGIERHI\SRNYRGXMSREREP]WMW

The BaseScope™ Assay 8[SEPXIVREXMZIGSRƤKYVEXMSRWSJXLMWOMXEVIEZEMPEFPIXSIREFPI JYPP]EYXSQEXIH[EPOE[E]-7,WSPYXMSRW 8LI&EWI7GSTIƎ%WWE]MWEYRMUYITVSHYGXJVSQ%'(FEWIH on the same platform of proven and established RNAscope® ŵ&EWI7GSTIƎ:76IEKIRX/MXJSVYWISRXLI(-7'3:)6= technology. This powerful assay provides new data ULTRA automated tissue staining systems by Roche Tissue dimensions and unique insight into biological mechanisms. Diagnostics. 8LI&EWI7GSTIƎ%WWE]IREFPIWETTPMGEXMSRWWYGLEWI\SR ŵ&EWI7GSTIƎ076IEKIRX/MXJSVYWISRXLI NYRGXMSRWJSVXLIEREP]WMWSJWTPMGIZEVMERXWHIXIGXMSRSJWLSVX Leica Biosystems’ BOND Rx System. RNA targets (50-300 bases), highly homologous sequences, For further information on our automated solutions please visit RNA mutations and, circular RNA, or gene fusions. acdbio.com/automated-assays With ACD’s continued commitment to provide researchers advanced capabilities of performing RNA ISH, the BaseScope™ Duplex Assay HIZIPSTQIRXSJXLI&EWI7GSTIƎTVSHYGXTVSZMHIWEOI] FIRIƤXXSHIXIGXFMSPSKMGEPIZIRXW in situ using a single Z pair 8LI&EWI7GSTIƎ(YTPI\%WWE]GERFIYWIHJSVWMQYPXERISYW MI62%WIUYIRGIWTERRMRKRX 8LI&EWI7GSTIƎEWWE] visualization of two RNA targets while maintaining single cell was developed with advances in probe design, leveraging core resolution. concepts of the ZZ patented technology, and applies a novel Applications include: EQTPMƤGEXMSRW]WXIQXLEXKIRIVEXIWMRGVIEWIHWMKREP[MXL • Discerning bi-allelic vs. mono-allelic CRISPR-mediated WMQYPXERISYWFEGOKVSYRHRSMWIWYTTVIWWMSR mutations • Co-detection of circRNAs and linear RNAs BaseScope™6IEKIRX/MXZ6)( • Simultaneous visualization of 2 splice variants or short The &EWI7GSTIƎ6IEKIRX/MXZ6)([MXLMXWLMKLGSRXVEWX Fast Red dye, uses alcaline phosphatase mediated detection targets with the FastRed substrate. ŵ 4VSƤPMRKI\TVIWWMSRSJEWTPMGIZEVMERXWLSVXXEVKIXSVKIRI IHMXMREGIPPWTIGMƤGQERRIVXEVKIXW

*-+96)(IXIGXMSRSJI\SRWOMTTIHZEVMERXSJ1)8Q62% 1)8± FIGURE 11. Visualization of CRISPR-mediated gene editing in the mouse liver in lung cancer cell line H596 using the &EWI7GSTIƎ6IEKIRX/MX6)( YWMRKXLI&EWI7GSTIƎ(YTPI\%WWE];MPHX]TIWIUYIRGIMRKVIIRIHMXIH sequence in red.

12 Advanced Cell Diagnostics RNAscope® in situ Manual Assay Featured Applications

Dual ISH-IHC RNAscope for Spatial Validation of Single-Cell Transcriptomes RNA in situ hybridization (ISH) and immunohistochemistry 8LI62%WGSTIoXIGLRSPSK]MWEVSFYWXLMKLP]WTIGMƤGERH (IHC) are well-established methods providing unique RNA sensitive RNA ISH methodology with multiplexing capabilities and protein expression with morphological context. Often to validate and provide spatial information for high-throughput considered complementary technologies, they bridge the single cell transcriptomic results. This technology allows for gap between RNA and protein analysis. By targeting different GIPPX]TIWTIGMƤGI\TVIWWMSRTVSƤPIWXSFIQETTIHFEGOXSXLI molecules, one the precursor of the other, performing ISH and complex tissue context of organs. IHC together can provide complementary information to: &IRIƤXWSJXLI62%WGSTIoXIGLRSPSK]MRGPYHI ŵ -HIRXMJ]XLISVMKMRSJWIGVIXIHTVSXIMRWũ-7,MHIRXMƤIWGIPPW ŵ :MWYEPGSRƤVQEXMSRSJMRHMZMHYEPKIRIERHKIRIWMKREXYVI

XLEXTVSHYGISJXLITVSXIMR-,'GIPPWXLEXXEOIYTXLI expression secreted protein • Single cell resolution of gene expression and co-localization • Identify complex tissue structure – in complex structures [MXLQYPXMTPIGIPPX]TIW-,'MHIRXMƤIWXLIGIPPX]TIERH-7, • Spatial localization of cell types and states in tissue detects RNA expression inside these cells environment • Identify regulation of gene expression - translational • Identifying proper single cells for quality transcriptome regulation controls can shut down protein synthesis, or analysis protein instability can render IHC ineffective. Analysis of both RNA and protein expression in the same tissue allows differentiation between inhibition of transcription and protein instability • Assess gene therapy – a combination of ISH and IHC can provide a useful comparison between levels of transduction and levels of protein. Successful transduction may not always translate to successful expression due to regulation or protein instability.

Alpi

Chga

CBC: Lgr5 Lgr5 Enterocytes: Alpi Enteroendocrine cells: Chga

FIGURE 12. Detection of CD4 (green), FOXP3 (yelow), IFNg (red) combined FIGURE 13. Single Cell Analysis - Simultaneous detection of 3 intestinal with an CD8 antibody (white) in lung cancer tissue using the RNAscope® LS cell populations. Here, Lgr5 (white), Alpi (green) and Chga (red) in mouse Multiplex Fluorescent Assay intestine.

RNAscope® Manual Solutions 13 RNAscope®HIXIGXMSROMXWIPIGXMSRKYMHI A RNA ISH solution for every need

® RNAscope®Ʉ,( RNAscope®Ʉ,( RNAscope Ʉ,( Assay-BROWN Assay-RED Duplex Assay Target mRNA >300 bases mRNA >300 bases mRNA >300 bases lncRNA > 300 bases lncRNA > 300 bases lncRNA > 300 bases Assay Type Chromogenic Chromogenic Chromogenic Dye Used Diaminobenzidine (DAB) Fast Red HRP-based Green and AP- based Fast Red

Probes Channel Designation Channel 1 (C1 Probes) Channel 1 (C1 Probes) Channel 1 & 2 (C1 & C2 Probes) Multiplexing Singleplex Singleplex Singleplex, Duplex /I]&IRIƤX Robust, sensitive, permanent Provides bright color stains with Utilizes 2 different staining stain. Most widely used LMKLGSRXVEWXXSFEGOKVSYRH enzymes therefore avoiding RNAscope assay ER]GVSWWXEPOFIX[IIRXLIX[S stains

Ideal For First time user Routine Studies of tissues with Studies interrogating two RNA applications IRHSKIRSYWGSPSVFEGOKVSYRH FMSQEVOIVWWMQYPXERISYWP] WYGLEWQIPERMRMRWOMRPMZIVSV PYRKWJVSQWQSOIVW

Novel Gene or +++++ +++++ +++ 9RORS[R)\TVIWWMSR Archival Specimens +++++ ++ +++ Microscope 7XERHEVHFVMKLXƤIPH 7XERHEVHFVMKLXƤIPH 7XERHEVHFVMKLXƤIPH Imaging System 1YPXMWTIGXVEPƥYSVIWGIRX imaging Sample Type FFPE Tissues FFPE Tissues FFPE Tissues Fixed Frozen Tissues Fixed Frozen Tissues Fixed Frozen Tissues Fresh Frozen Tissues Fresh Frozen Tissues Fresh Frozen Tissues Cultured cells Cultured cells Cultured cells

Assay protocol length 8 hours (with ~2 hours hands- 8 hours (with ~2 hours hands- 13 hours (with ~3 hours hands- (steps 1-3) on time) on time) on time)

14 Advanced Cell Diagnostics RNAscope® Mulitplex RNAscope® Multiplex BaseScope™ Reagent BaseScope™ Duplex Fluorescent Assay *PYSVIWGIRXZ /MXZ6)( Assay Target mRNA >300 bases mRNA >300 bases RNA 50 to 300 bases RNA 50 to 300 bases lncRNA > 300 bases lncRNA > 300 bases )\SRNYRGXMSR )\SRNYRGXMSR Assay Type Fluorescent Fluorescent Chromogenic Chromogenic Dye Used FITC, Cy3, Cy5, Cy7 4IVOMR)PQIV Fast Red HRP-based Green and - PN NEL741001KT AP-based Fast Red - PN NEL744001KT - NEL745001KT 3TEPƎ(]IW - Part No. FP1487001KT - Part No. FP1488001KT - Part No. FP1495001KT - Part No. FP1497001KTP Probes Channel Channel 1-3 Channel 1-4 Channel 1 (C1 Probes) Channel 1 & 2 Designation (C1, C2 & C3 Probes) (C1, C2, C3 & C4 Probes) (C1 & C2 Probes) Multiplexing Single to Triplex Single to 4-plex Singleplex Singleplex, Duplex /I]&IRIƤX Utilizes up to 3 different TSA-based Fluorescent Provides bright color Utilizes 2 different spectral channels RNAscope® detection stains with high contrast staining enzymes assay for up to 4-plex TVSZMHMRKLMKLƥI\MFMPMX] XSFEGOKVSYRH therefore avoiding any capability using TSA ƥYSVSTLSVIW WSPH GVSWWXEPOFIX[IIRXLI separately). Ideal for two stains FFPE tissues

Ideal For Co-expression Co-expression circRNA, splice variants, circRNA, splice studies of up to 3 studies of up to 4 short targets, gene variants, genes simultaneously genes simultaneously editing short targets, gene Experimental application Experimental application editing, Studies VIUYMVMRKƥI\MFMPMX] VIUYMVMRKƥI\MFMPMX]**4) interrogating two tissue 62%FMSQEVOIVW simultaneously Novel Gene or +++ ++++ +++++ +++ 9RORS[R)\TVIWWMSR Archival Specimens ++ ++++ ++ +++ Microscope Multispectral 1YPXMWTIGXVEPƥYSVIWGIRX Standard bright 7XERHEVHFVMKLXƤIPH Imaging System ƥYSVIWGIRXMQEKMRK imaging ƤIPH1YPXMWTIGXVEP ƥYSVIWGIRXMQEKMRK Sample Type Fresh Frozen Tissues FFPE Tissues FFPE cells & tissues FFPE cells & tissues Cultured cells Fixed Frozen Tissues Fresh frozen tissues Fresh frozen tissues Fixed Frozen Tissues Fresh Frozen Tissues Cultured cells Cultured cells Cultured cells

Assay protocol length 6.5 hours (with ~2 hours 14 hrs (with ~5 hours 10 hours (with ~2 hours 12 hours (with ~3 hours (steps 1-3) hands-on time) hands-on time, can split hands-on time , can split hands-on time , can over 2 days) over 2 days) split over 2 days)

RNAscope® Manual Solutions 15 RNAscope® accessories Optimal temperature and humidity for optimal assay performance

HybEZ™ II Hybridization System Processing System Successful implementation of RNAscope® Assay is directly The ACD EZ-BatchƎ Slide Processing System facilitates PMROIHXSL]FVMHM^EXMSRIRZMVSRQIRX8LI%'(,]F)>Ǝ II processing multiple sample slides simultaneously. We ,]FVMHM^EXMSR7]WXIQERHMXWEFMPMX]XSEGGYVEXIP]OIITXLI HIWMKRIHXLMWTVSHYGXJSVLMKLIVIJƤGMIRG]MRVYRRMRKXLI temperature stable is essential to the success of RNAscope® manual RNAscope® assay protocol. The system comprises and BaseScopeƎ[SVOƥS[W RNAscope® EZ-BatchƎ Slide Holder and EZBatchƎ Wash Tray. The HybEZƎ--3ZIRMWEWMQTPIIEW]XSYWIPS[TVSƤPI The ACD EZ-BatchƎ Slide Holder is fully compatible with the benchtop hybridization oven that provides superior ACD HybEZƎ Humidity Control Tray and is designed with conditions for RNA-ISH, and is the only hybridization oven ERIEW]PSGOMRKQIGLERMWQXSOIITWPMHIWMRXEGXHYVMRK that guarantees optial performances of the RNAscope® washing steps. This design eliminates the time-consuming and BaseScopeƎ assays. The HybEZƎ II Oven provides a XVERWJIVSJWPMHIWFIX[IIRXLIWPMHIVEGOERH8MWWYI8IO KEWOIXWIEPIHXIQTIVEXYVIGSRXVSPPIHLYQMHMJ]MRKGLEQFIV washing tray during wash steps and ensure optimal washing necessary for optimized RNAscope® assay performance. IJƤGMIRG])EGL%'()>&EXGLƎ Slide Processing System can accommodate 20 slides. This instrument system is capable of holding 20 slides at a set temperature and high humidity for hybridization and other 7GMIRXMWXWERHPEFXIGLRMGMERWVSYXMRIP]IQTPS]XLMWIJƤGMIRX MRGYFEXMSRWXITWEWWTIGMƤIHMRXLIQERYEP62%WGSTI® and tool to RNAscope® Assay and also to other protocols where BaseScopeƎ protocols. handling multiple slides is required. Why handle one slide at a time when you can process all 20 at once? 8LIW]WXIQGSQTVMWIW ƤKYVI ,]F)>SZIR 42 321710/321720), a humidity control tray (PN 310012), and HybEZ Humidifying Paper (2 sheets PN 310025), EZ-Batch Wash Tray (PN 310019), EZ-Batch Slide Holder (PN 310017)

RNAscope® EZ-Batch™ Slide

FIGURE 14. ,]F)>Ǝ--,]FVMHM^EXMSR7]WXIQ

16 Advanced Cell Diagnostics RNAscope® in situ Manual Assay ;SVOƥS[ERHEWWSGMEXIHTVSHYGXW GSRXMRYIH

Step 04. View your results Visualize

Each punctate dot signal represents a single target RNA molecule and can be visualized with a standard microscope ƤKYVI )\EQMRIXMWWYIWIGXMSRWYRHIVEWXERHEVHFVMKLXƤIPH QMGVSWGSTISVWXERHEVHƥYSVIWGIRXQMGVSWGSTIEXũ< FIGURE 15. PPIB detection (brown punctate dots) in human cervix using QEKRMƤGEXMSRSV[MXLEQYPXMWTIGXVEPƥYSVIWGIRXMQEKMRK RNAscope®,(OMX&63;2Each brown punctate dot signal represents microscope to: a single target RNA molecule and the size of the dot is proportional to the number of double Z probes hybridized on the target RNA molecule. • Assess tissue and cell morphology and quality. • Assess positive control signal strength. Positive control WMKREPWLSYPHFIZMWMFPIEWTYRGXYEXIHSXWEXũ< QEKRMƤGEXMSR ŵ%WWIWWRIKEXMZIGSRXVSPFEGOKVSYRH3RIHSXXSIZIV] GIPPWHMWTPE]MRKFEGOKVSYRHDapBWXEMRMRKTIV< QMGVSWGSTIƤIPHMWEGGITXEFPI • Assess differences between single dots versus dot clusters. 8LMWMWMQTSVXERXJSVWGSVMRKJSVUYERXMƤGEXMSR

7MRKPITPI\WIQMUYERXMXEXMZIWGSVMRK :MWYEPWGSVMRKMWTIVJSVQIHEX\QEKRMƤGEXMSRXSEWWMKRE single score to a sample based on the predominant staining pattern seen throughout the entire sample. Intensity of the stain does not have an impact on scoring. Dots correlate to FIGURE 16. Singleplex semi-quantitative scoring the number of individual RNA molecules, whereas dot intensity VIƥIGXWXLIRYQFIVSJTVSFITEMVWFSYRHXSIEGLQSPIGYPI Percentage of cells positive is scored visually based on number SJGIPPW[MXLƞHSXGIPPERHFMRRIHMRXSGEXIKSVMIW

Visual H-scoring 8SIZEPYEXILIXIVSKIRIMX]MRQEVOIVI\TVIWWMSR,WGSVI calculation is performed and a single score is assigned to a region of interest (ROI). Cells are grouped into 5 bins based on the number of dots per cell. Clusters are divided by the typical probe signal area to calculate a dot number for the cluster. Each sample is evaluated for the percentage of cells in each bin. The H-score is calculated by totaling the percentage of cells in each bin according to the weighted formula shown. H-scores are typically provided on a scale of 0–400.

FIGURE 17. Visual H-scoring

For more information request or download the RNAscope® Data Analysis Guide containing data analysis guidelines for several types of RNAscope® staining results. acdbio.com/dataanalysisguide

RNAscope® Manual Solutions 17 RNAscope® in situ Manual Assay ;SVOƥS[ERHEWWSGMEXIHTVSHYGXW GSRXMRYIH

Step 05. 7SJX[EVIJSVUYERXMXMZIEREP]WMW Quantify %GGYVEXIUYERXMƤGEXMSREX]SYVƤRKIVXMTW

The single-molecule sensitivity and visualization of the analysis software. It is an intuitive automated solution that RNAscope®XIGLRSPSK]QEOIWUYERXMXEXMZI62%in situ KIRIVEXIWWXERHEVHM^IHERHSFNIGXMZIVIWYPXWMRQMRYXIW TM hybridization analysis a reality. HALO Software can be used to analyze data generated with GLVSQSKIRMGHYTPI\GLVSQSKIRMGERHƥYSVIWGIRX62%WGSTI® ACD as partnered with Indica Labs to facilitate and improve Assays. HALOTM Software is commercialized by Indica Labs. quantitative scoring by providing software for automated analysis – HALOTM Software. This advanced image analysis An additional collaboration with Leica Biosystems enables WSPYXMSRFVMRKWSFNIGXMZIERHEGGYVEXIUYERXMƤGEXMSRXS62% VIEWIEVGLIVWYWMRKXLI0IMGE&MSW]WXIQW&32(6

FIGURE 18. PPIB expression analysis in human breast carcinoma sample using HALOTM software (Indica Labs).

FIGURE 19.Software-based image image analysis is performed using HALOTM software (Indica Labs).

18 Advanced Cell Diagnostics ACD Support Scientists offer ISH expertise to ensure your success

'SQTVILIRWMZI[SVPH[MHIWYTTSVX Field support and onsite trainings Whether you need help selecting your target or deciding which We pride ourselves in advancing our client’s research by RNAscope® assay format best suits your needs, our Global SJJIVMRKXLIFIWXWGMIRXMƤGWYTTSVXSVKYMHERGIIMXLIVZME Technical and Field Support Scientists are here to assist you TLSRIIQEMPSVSRWMXIZMWMXW-J]SY[SYPHPMOISYVWYTTSVX through the process of selecting your assay, designing your scientists to visit your lab and provide training on site, please experiments, and guiding you through the interpretation for a contact your sales or account executive. A Field Application successful RNAscope® research analysis. Scientist will visit you shortly and provide an onsite training to you and members of your lab. New user program %W]SYFIKMR]SYVƤVWXRNAscope® assay, we offer an Contact us exclusive new user program, providing guidance for every North America ACD Support Scientists are available between step from experimental design and set up, to interpretation EQXSTQ4EGMƤG7XERHEVH8MQI of results. With our support scientists’ assistance, we are GSRƤHIRX]SYGERSFXEMRTYFPMGEXMSRUYEPMX]VIWYPXWJVSQ ]SYVƤVWXEWWE];IEPWSSJJIVQSRXLP]XIGLRMGEPWYTTSVX webinars for new or existing users with details on our manual GLVSQSKIRMGƥYSVIWGIRXERHSVEYXSQEXIHEWWE]TVSGIHYVIW =SYGEREP[E]W[EXGLWYTTSVXZMHISWERHVIGSVHIH[IFMREVW Email Support: [email protected] available on our website at acdbio.com/learn-more Phone Contact: 1-877-576-3636

In Europe, ACD Support Scientists are available between 8:00 am to 6:00 pm Central European Time

Email Support: [email protected]

Phone Contact: +44 1235 529 449

RNAscope® Manual Solutions 19 4VSHYGXSZIVZMI[

Chromogenic Singleplex Chromogenic Fluorescent Brown Singleplex Red Duplex Multiplex

RNAscope® Assay Manual assays

Automated on Leica BOND RX System Automated on DISCOVERY ULTRA automated tissue staining systems by Roche Tissue Diagnostics BaseScope™ Assay Manual assays

Automated on Leica BOND RX System Automated on DISCOVERY ULTRA and DISCOVERY XT* automated tissue staining systems by Roche Tissue Diagnostics Pharma Assay Manual assays 7IVZMGIW Automated on Leica BOND RX System Automated on DISCOVERY ULTRA automated tissue staining systems by Roche Tissue Diagnostics

Learn more about the RNAscope® assay at acdbio.com

*SV6IWIEVGL9WI3RP]2SXJSVHMEKRSWXMGYWI62%WGSTIMWEVIKMWXIVIHXVEHIQEVOSJ Advanced Cell Diagnostics, Inc. in the United States or other countries. All rights reserved. ©2018 Advanced Cell Diagnostics, Inc. Doc #: MK 51-064/Rev.C/Effective Date 01282019 California, USA

20 Advanced Cell Diagnostics