In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays. In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays. Biomarkers based on DNA, RNA, and proteins are increasingly used for cancer diagnosis, prognosis, and therapy guidance, heralding the era of personalized medicine.1Hamburg M.A. Collins F.S. The path to personalized medicine [Erratum appeared in N Engl J Med 2010;363:1092].N Engl J Med. 2010; 363: 301-304Crossref PubMed Scopus (1348) Google Scholar RNA biomarkers or gene expression signatures have emerged as a major class of cancer biomarkers, thanks to widespread use of genome-wide gene expression profiling technologies.2Sotiriou C. Piccart M.J. Taking gene-expression profiling to the clinic: when will molecular signatures become relevant to patient care?.Nat Rev Cancer. 2007; 7: 545-553Crossref PubMed Scopus (397) Google Scholar, 3van 't Veer L.J. Dai H. van de Vijver M.J. He Y.D. Hart A.A. Mao M. Peterse H.L. van der Kooy K. Marton M.J. Witteveen A.T. Schreiber G.J. Kerkhoven R.M. Roberts C. Linsley P.S. Bernards R. Friend S.H. Gene expression profiling predicts clinical outcome of breast cancer.Nature. 2002; 415: 530-536Crossref PubMed Scopus (7723) Google Scholar To implement these genomic signatures in clinical diagnostic assays, the current platform of choice is real-time RT-PCR, which is considered the gold standard in gene expression analysis.4Wong M.L. Medrano J.F. Real-time PCR for mRNA quantitation.Biotechniques. 2005; 39: 75-85Crossref PubMed Scopus (1258) Google Scholar However, this grind-and-bind approach has a serious drawback: the process of RNA extraction destroys the tissue context of gene expression measurements, making it impossible to map the observed signals to individual cells. Furthermore, these assays are prone to interference from unintended cell types (eg, noncancer cells) and from unwanted tissue elements (eg, fibrosis and necrosis). Microdissection techniques can alleviate this problem to some extent,5Emmert-Buck M.R. Bonner R.F. Smith P.D. Chuaqui R.F. Zhuang Z. Goldstein S.R. Weiss R.A. Liotta L.A. Laser capture microdissection.Science. 1996; 274: 998-1001Crossref PubMed Scopus (2121) Google Scholar, 6Bonner R.F. Emmert-Buck M. Cole K. Pohida T. Chuaqui R. Goldstein S. Liotta L.A. Laser capture microdissection: molecular analysis of tissue.Science. 1997; 278 (1481, 1483)Crossref PubMed Scopus (781) Google Scholar but they are too cumbersome and laborious to be useful on a routine basis. By contrast, DNA in situ hybridization (ISH)7Levsky J.M. Singer R.H. Fluorescence in situ hybridization: past, present and future.J Cell Sci. 2003; 116: 2833-2838Crossref PubMed Scopus (380) Google Scholar and protein immunohistochemistry (IHC)8Matos LL de Trufelli D.C. de Matos M.G.L. da Silva Pinhal M.A. Immunohistochemistry as an important tool in biomarkers detection and clinical practice.Biomark Insights. 2010; 5: 9-20Crossref PubMed Google Scholar are routinely used in clinical laboratories for DNA and protein biomarker analysis, allowing the integration of molecular information with histopathology for optimal clinical interpretation. To date, use of RNA ISH in clinical diagnostics has been limited to highly expressed genes such as Epstein-Barr virus (EBV)-derived transcripts EBER1/2 in EBV-related diseases.9Gulley M.L. Molecular diagnosis of Epstein-Barr virus-related diseases.J Mol Diagn. 2001; 3: 1-10Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 10Ambinder R.F. Mann R.B. Detection and characterization of Epstein-Barr virus in clinical specimens.Am J Pathol. 1994; 145: 239-252PubMed Google Scholar Conventional non-radioisotopic RNA in situ hybridization (ISH) techniques lack the sensitivity and specificity required to measure many low-abundance RNA biomarkers reliably.11Mahmood R. Mason I. In-situ hybridization of radioactive riboprobes to RNA in tissue sections.Methods Mol Biol. 2008; 461: 675-686Crossref PubMed Scopus (12) Google Scholar Here, we describe RNAscope, a novel RNA ISH method. Single-molecule visualization in individual cells is achieved through use of a novel probe design strategy and a hybridization-based signal amplification system to simultaneously amplify signals and suppress background. Many of the steps in RNAscope are similar to those in IHC. The RNAscope approach can be used with archival formalin-fixed, paraffin-embedded (FFPE) tissue samples on glass slides, and the stained slides can be visualized under either a standard bright-field microscope (with chromogenic labels) or an epifluorescent microscope (with fluorescent labels). The RNAscope approach allows multiplex detection for up to four target genes (limited by the number of spectrally discernible fluorescent dyes). The ability to analyze gene expression in situ in routine clinical specimen types, as well as high sensitivity and specificity, make RNAscope a promising platform for translating many RNA biomarkers into clinical use. SK-BR-3 breast adenocarcinoma cells (ATCC, Manassas, VA) were cultured in McCoy's medium. HuH-7 hepatocellular carcinoma cells (JCRB-Japanese Collection of Research Bioresources, Shinjuku, Japan) with or without hepatitis C virus (HCV) infection were cultured in modified Eagle's medium. MCF7 breast adenocarcinoma cells, SiHa cervical squamous cell carcinoma cells, HeLa cervical adenocarcinoma cells, and MS751 cervical epidermoid carcinoma cells (all from ATCC) were cultured in Dulbecco's modified Eagle's medium at 37°C in 5% CO2. MDA-MB-468 breast adenocarcinoma cells (ATCC) were cultured in L-15 (Leibovitz) medium in a CO2-free incubator at 37°C. All media were supplemented with 10% fetal bovine serum (Gibco; Invitrogen, Carlsbad, CA). Deidentified archival FFPE tumor tissues were purchased from Analytical Biological Services (Wilmington, DE). Tissue quality was assessed by performing RNAscope analysis for mRNA of the housekeeping gene ubiquitin C (UBC). We sought to improve the signal-to-noise ratio of RNA ISH by amplifying target-specific signals but not background noise from nonspecific hybridization. We used a novel target probe design strategy (a double-Z design) (Figure 1). A series of target probes are designed to hybridize to the target RNA molecule. Each target probe contains an 18- to 25-base region complementary to the target RNA, a spacer sequence, and a 14-base tail sequence (conceptualized as Z). A pair of target probes (double Z), each possessing a different type of tail sequence, hybridize contiguously to a target region (∼50 bases). The two tail sequences together form a 28-base hybridization site for the preamplifier, which contains 20 binding sites for the amplifier, which, in turn, contains 20 binding sites for the label probe. Typically, a 1-kb region on the RNA molecule is targeted by 20 probe pairs; thus, sequential hybridizations with the preamplifier, amplifier, and label probe can theoretically yield up to 8000 labels for each target RNA molecule. This hybridization-mediated signal amplification scheme is similar to the branched DNA (bDNA) method described previously,12Player A.N. Shen L.P. Kenny D. Antao V.P. Kolberg J.A. Single-copy gene detection using branched DNA (bDNA) in situ hybridization.J Histochem Cytochem. 2001; 49: 603-612Crossref PubMed Scopus (136) Google Scholar but the double-Z probe design strategy should ensure superior background control because it is highly unlikely that a nonspecific hybridization event will juxtapose a pair of target probes along an off-target mRNA molecule to form the 28-base hybridization site for the preamplifier, and also because a single 14-base tail sequence will not bind the preamplifier with sufficient strength to result in successful signal amplification. The label probe can be either fluorescently labeled for direct visualization under an epifluorescent microscope or conjugated to an alkaline phosphatase or horseradish peroxidase (HRP) molecule for chromogenic reactions [Fast Red with alkaline phosphatase and 3,3′-diaminobenzidine (DAB) with HRP]. The alkaline phosphatase or HRP-labeled probes have an added advantage, in that chromogen-stained slides can be viewed under a standard bright-field microscope similar to IHC procedures, making RNAscope assay results easier to read and archive in a clinical setting. Multiple RNA species can be measured simultaneously in two ways: the target probes for different genes can have the same tail sequence recognized by the same signal amplification system, generating a pooled signal; alternatively, multiple signal amplification systems with different label probes can be used to detect each RNA species, allowing for multiplex detection of multiple target RNAs. Custom software was written to automatically select target probe sequences with compatible melting temperature (Tm) and minimal cross-hybridization to off-target sequences.13Bushnell S. Budde J. Catino T. Cole J. Derti A. Kelso R. Collins M.L. Molino G. Sheridan P. Monahan J. Urdea M. ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.Bioinformatics. 1999; 15: 348-355Crossref PubMed Scopus (30) Google Scholar We determined that three probe pairs could generate readily visible signals with HRP/DAB detection (see Supplemental Figure S1 at ). We chose 10 to 20 pairs, for optimal signals and for added robustness against potentially variable target accessibility and partial RNA degradation. The target genes and probed regions are listed in Supplemental Table S1 (available at ). Sequences of target probes, preamplifier, amplifier, and label probe are proprietary (Advanced Cell Diagnostics, Hayward, CA). For fluorescent detection, the label probe was conjugated to Alexa Fluor 488, 546, 647, or 750 (Molecular Probes; Invitrogen, Eugene, OR). For chromogenic detection using DAB, label probe was conjugated to HRP. For cell lines, cells were placed on slides and fixed in 4% formaldehyde for 60 minutes, followed by protease digestion (2.5 μg/mL) at 23°C to 25°C. The cells were then incubated in order at 40°C with the following solutions: target probes in hybridization buffer A [6× SSC (1× SSC is 0.15 for preamplifier in hybridization buffer 10% for in hybridization buffer at 40°C for and label probe in hybridization buffer C for each hybridization slides were with buffer three at For multiplex detection, of target probes, preamplifier, amplifier, and label probe of each amplification system were detection was using followed by with CA). For FFPE tissue in were in followed by in an Tissue were then incubated in buffer at a temperature to using a for minutes, in and protease at 40°C for in a hybridization (Advanced Cell Diagnostics, Hayward, CA). with target probes, preamplifier, amplifier, and label probe and chromogenic detection were as described for cultured cells. A of assay were to for FFPE samples and fixed to of of M. D.C. S. G. Goldstein M. S. R. K. S. J. A. H. S. P. G. D. T. R.B. of of for of and in breast Med. 2010; appeared in Med Google Scholar for to at such as buffer and protease using archival FFPE specimens were in with and to ensure The housekeeping gene was used as control to assess tissue RNA and assay with signals visible under a was considered to be The gene was used as control to assess background was routinely achieved using standard A was used to detect mRNA in tissue to the in with the RNAscope control was in the cells were in a fixed in and on The cells were then with Diagnostics, followed by for 10 at 37°C. DNA was in for at and then was to probe hybridization to the RNAscope described HeLa cells were cultured in was to a cell was for analysis to the the was used for RNAscope analysis. The number of mRNA transcripts in the cell was determined by using a standard from of in The number was determined by the number of mRNA in the by the number of cells used in the were by based on The number of mRNA transcripts determined by RNAscope was based on the fluorescent of cells in The number was determined by the by the number of cells were using an fluorescent microscope Japan) and a For multiplex RNAscope and for with FFPE tissue were using a microscope and a system signals from different were by signals against a with stained We that amplification of target-specific signals without amplifying background signal should improve signal-to-noise also sensitivity and specificity in RNA To achieve this we a probe design to in which two probes (double have to hybridize to the target sequence in in order for signal amplification to (Figure also under and it is highly unlikely that two probes will hybridize to a nonspecific target to each this design should in ensure amplification of target-specific To this probe design we determined the target probes are to generate The of target probe pairs for RNA was into two of the probes with the tail and the of the probes with the tail (Figure The two of probes were either or in with HeLa cells. A fluorescent was observed when were used but fluorescent signal was when either was used The lack of visible signals from the control with target from either of the target probe pairs was considering the high of target in the cells. This result that the individual 14-base were not sufficient for binding to the signal amplification system under the assay To assay specificity, a probe RNA was used to detect a in HuH-7 cells with was in cells, but not in control cells (Figure To the of specificity we stained cell different with as as sequence H. and from to clinical Rev Cancer. 2002; PubMed Scopus Google Scholar Each target probe in the cell to the (see Supplemental Figure at ). RNAscope, a probe hybridization-based could have an IHC assay because the assay is to RNA transcripts are within the and because cross-hybridization target probes can be by the probe design To the ability of RNAscope to detect multiple transcripts probe for the housekeeping genes and were labeled with fluorescent dyes of different and to HeLa cells (Figure All four genes expression with and high expression To multiplex analysis results similar to those of analysis, we of and in SK-BR-3 cells either or in a The for each gene were similar for single and hybridization (see Supplemental Table at ). The for and the that each fluorescent in the RNAscope assay a single mRNA We used two different methods to we used the same target probes to detect mRNA and genomic DNA in HeLa and SK-BR-3 cells under target probe hybridization and signal amplification For genomic DNA, two fluorescent were in HeLa cells, many were in SK-BR-3 cells, with the and gene status in HeLa and SK-BR-3 cells, (Figure results that the RNAscope assay is of single-molecule The same probe mRNA transcripts in HeLa and SK-BR-3 cells with with gene amplification status (see Supplemental Figure at ). We then the signal of mRNA with those of genomic DNA the RNA mRNA then the signal of at some of the mRNA be to be the genomic DNA However, mRNA signal a fluorescent the observed for genomic DNA (see Supplemental Figure at ). In the signal of the RNA was the because of the probe accessibility or of In a we the number of mRNA in HeLa cells and that with the number of mRNA transcripts determined in cell by C. K. A. K. of DNA results with gene expression PubMed Scopus Google Scholar (Figure and The number cell determined by RNAscope Figure well with the number cell determined by Figure these results are with each RNA from a single mRNA molecule. FFPE tissue is the widely used clinical type in cancer diagnosis, we the RNAscope assay for use on FFPE tissue and determined for tissue A probe the housekeeping gene was to archival and FFPE using standard tissue in buffer and protease label was in three tissue types, but signal was present when the control probes were used (Figure To the detection sensitivity of RNAscope in FFPE we probe for two housekeeping and to breast tumor signals were observed for genes as fluorescent (Figure which are to those in cell lines, the of single-molecule We RNAscope with a non-radioisotopic RNA ISH used to the for expression in in FFPE cells in the high of cells in the of that be by conventional non-radioisotopic ISH In situ hybridization analysis of of by RNA Mol Pathol. 1994; 3: PubMed Scopus Google Scholar, M. J. in situ hybridization detection of mRNA expression in cell and Mol Pathol. 2003; PubMed Scopus Google Scholar RNAscope and in cells, RNAscope was RNAscope in cells in the a in these cells (Figure a that can be to from M. J. in situ hybridization detection of mRNA expression in cell and Mol Pathol. 2003; PubMed Scopus Google Scholar To the multiplex ability of RNAscope in FFPE we the expression of and in FFPE breast cancer together with probes for and using a assay (Figure and are the in breast H. R. P. S. M.R. of of of for the use of tumor in breast 2007; PubMed Scopus Google Scholar, K. M. M. D. S. G. C. and in breast of clinical and current in the 2008; PubMed Scopus Google Scholar with R. J.M. C. C. L.A. A. of type and type and protein and mRNA in breast 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. S. J. M. K. is in cells in breast 2001; PubMed Scopus Google Scholar and mRNA was in cells, but not in the tumor Furthermore, results that and are in the same that these two proteins may be by different cell M. S. S. J. M. K. is in cells in breast 2001; PubMed Scopus Google Scholar Here, we have described the development and of RNAscope, a novel non-radioisotopic RNA ISH technology that has the to be to routine clinical samples for biomarker analysis. which has a of has been J.M. Singer R.H. Fluorescence in situ hybridization: past, present and future.J Cell Sci. 2003; 116: 2833-2838Crossref PubMed Scopus (380) Google Scholar, in signal amplification methods for in situ Mol Pathol. 2003; PubMed Scopus Google Scholar, A. van P. van A. S. individual mRNA using multiple labeled 2008; 5: PubMed Scopus Google Scholar, of single RNA transcripts in PubMed Scopus Google Scholar in sensitivity and specificity and the have in clinical and at RNA ISH have largely on J.M. Singer R.H. Fluorescence in situ hybridization: past, present and future.J Cell Sci. 2003; 116: 2833-2838Crossref PubMed Scopus (380) Google Scholar, in signal amplification methods for in situ Mol Pathol. 2003; PubMed Scopus Google Scholar either by amplifying the mRNA hybridization (eg, in situ G.J. In situ and PubMed Google or amplifying the signals target hybridization (eg, A.N. Shen L.P. Kenny D. Antao V.P. Kolberg J.A. Single-copy gene detection using branched DNA (bDNA) in situ hybridization.J Histochem Cytochem. 2001; 49: 603-612Crossref PubMed Scopus (136) Google Scholar or signal P. J. P. mRNA in situ hybridization on and paraffin-embedded tissue using signal amplification with different Cell Biol. PubMed Scopus Google amplification hybridization because of amplification and signal amplification can also amplify the in signal-to-noise In contrast, RNAscope was designed to amplify target-specific signals without also amplifying the in in signal-to-noise To date, we have RNAscope assays for genes with expression In biomarkers in FFPE such as from tissue to and can the biomarker M. D.C. S. G. Goldstein M. S. R. K. S. J. A. H. S. P. G. D. T. R.B. of of for of and in breast Med. 2010; appeared in Med Google Scholar We that results could be tissues were and fixed to M. D.C. S. G. Goldstein M. S. R. K. S. J. A. H. S. P. G. D. T. R.B. of of for of and in breast Med. 2010; appeared in Med Google Scholar The use of up to 20 target probe pairs, each to along the target RNA should robustness against the partial RNA of FFPE proteins such as and proteins are increasingly used as and diagnostic In in which these proteins by IHC may lack sensitivity or for M. J. in situ hybridization detection of mRNA expression in cell and Mol Pathol. 2003; PubMed Scopus Google by RNAscope may be an because are In RNAscope a in RNA ISH and is compatible with clinical types and has to this assay in routine clinical samples from clinical mRNA detection by a novel hybridization assay with expression and patient in squamous cell J Pathol. PubMed Scopus Google Scholar the target probes are and can be designed and RNAscope assays should be to with and assay in situ such as DNA ISH and protein RNAscope as a platform for and molecular We and Urdea for and and for and in assay and for HuH-7 and cell with Supplemental Figure of probe pairs required for generating visible signal in chromogenic RNAscope HeLa cell FFPE was used as a system for the number of probe pairs to detect in FFPE tissue specimens. was with the probe of 20 target probe pairs or with probe as were with The probe was into 20 individual target probe pairs, to 20 based on along the target to was then using target pair or a of two target pairs 10 and or a of three target pairs at the of the probe and were also using or three target pairs at the or of the probe similar results were with Supplemental Figure of from three cervical cancer cell to different SiHa HeLa and MS751 were stained by RNAscope with probe and was with an alkaline phosphatase label probe followed by with Red which was visualized under a were with with Supplemental Figure mRNA detection in HeLa cells. The same fluorescently labeled probes and signal amplification system were used to detect genomic DNA (see Figure and mRNA in HeLa and SK-BR-3 cells. signal of individual fluorescent in HeLa cells were using analysis software for and cell and were as for RNA and with Supplemental Table S1 with Supplemental Table