WISH you could get results like these from your tissue samples?

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1 Olfactomedin (Olfm4, red) in crypt base stem cells in mouse colon with Gapd housekeeping control (blue) WISH you could get results like these from your tissue samples? ViewRNA Assays. Find out more.

2 WISH you had a partner to help you all the way? Ask Affy. Affymetrix is your partner from bench to bedside With ViewRNA Assays for RNA in situ hybridization (ISH), you re getting more than just reagents. You re getting a collaborator who understands what you need to develop your assays from early research all the way to validation. Affymetrix is committed to accelerating RNA-based discovery through the entire molecular pathology spectrum including translational research, pharmaceutical discovery and development, and clinical assay development. GPR- and ASR-grade reagents Medical and regulatory affairs specialists in-house Expert molecular pathologists and bioinformaticians in-house A partnership with Affymetrix brings unmatched expertise Whole-genome to single-rna analysis RUO to IVD Expert, collaborative customer support Expert probe design and bioinformatics Quality and consistency Alpha-methylacyl coenzyme A racemase (AMACR; red) in human prostatic adenocarcinoma. 2

3 The power of ViewRNA Assays ViewRNA Assays bring a new level of power and simplicity to RNA ISH, enabling you to visualize targets that may not be easily localized using other technologies. Secreted proteins Targets for which antibodies are not readily available Long non-coding RNAs ViewRNA Assays rely on a completely different technology than immunohistochemistry (IHC); based on nucleic acids rather than proteins, they offer a variety of advantages: No need to develop antibodies Reproducible probe synthesis Custom probes shipped within a week Over 6,000 catalog probes available Uncompromising performance ViewRNA Assays components are developed to work together to achieve the highest possible signal, lowest background, and most stringent specificity. Unhindered by crosslinking Probes readily hybridize, even to partially degraded target RNA sequences Single-RNA-molecule sensitivity, detectable at single-cell resolution Single-molecule sensitivity Works with many tissues Formalin-fixed paraffin-embedded (FFPE) Any tissue ViewRNA technology Exquisite specificity Fresh-frozen Tissue microarrays (TMAs) Fine-needle aspirates Probes Buffers Signal amplifiers Chromogens Any RNA target Low background 3

4 WISH you could go where IHC can t? ViewRNA Assays enabling new insights and better-informed decision making Localize secreted protein transcripts Localization of secreted proteins can be challenging due to the wide distribution of the secreted target protein throughout the tissue. However, the RNA transcripts are not secreted and can be localized at the cellular level using ViewRNA Assays. These assays can be run in either manual or automated formats, simplifying biomarker validation with high-throughput workflows. Studies at Massachusetts General Hospital and Harvard Medical School found 100% concordance between albumin mrna ISH assays run both manually and on Leica Biosystems BOND RX (data not shown). 1 A B (A) In retrospective studies on archived samples of known outcome, FFPE TMAs stained with ViewRNA ez Assays on Leica Biosystems BOND RX revealed differential localization of albumin transcripts (red) on cores from confirmed cases of hepatocellular carcinoma, normal liver and metastatic adenocarcinoma. (B) A magnified image of metastatic adenocarcinoma shows the high specificity of the assays with no signal for albumin (as expected). A B C N HCC Normal liver Hepatocellular carcinoma (HCC) Metastatic adenocarcinoma (secondary liver cancer) The TMA studies above were followed by retrospective studies on archived FFPE tissue sections of known outcome, also stained with ViewRNA ez Assays on Leica Biosystems BOND RX. Normal liver (A) shows high levels of albumin expression (red) in hepatocytes, with no albumin expression visible in the interspersed bile ductules. Likewise (B), a primary liver cancer (hepatocellular carcinoma, HCC) and adjacent normal liver (N) also both show high levels of albumin expression, while interspersed stromal tissue is negative for albumin. In contrast (C), a secondary liver cancer (metastatic adenocarcinoma) shows no albumin expression. 4

5 Reveal targets in pigmented tissues Wnt proteins form a family of highly conserved signalling molecules with complex regulatory roles in processes including embryonic development, diabetes, and cancer progression. Particularly in melanoma, visualization of Wnt proteins can be especially challenging, both because the proteins are secreted and because the brown melanin pigment can mask detection stains. With ViewRNA Assays, Wnt RNA transcripts are clearly localized with the Fast Red signal that is easily distinguished from melanin. A B (A) Distribution of Wnt transcripts (red) in mouse skin hair follicles. (B) Magnified view reveals Fast Red signal dots indicating individual Wnt mrna transcripts (arrowheads), along with brown-pigmented melanocytes (long arrows). Visualize non-coding RNAs Circular RNAs (circrnas) are a class of low-abundance cellular RNAs with no 5 or 3 end. Neuronal circrnas are composed of protein-coding exons, but do not appear to be translated into peptides. With no poly-a tail and no protein, how do you even begin to find them? A team at the Berlin Institute for Medical Systems Biology, Max Delbrueck Center, and Max Planck Institute believe they may be the first ever to have visualized individual circrna species directly in mouse hippocampus, enabling them to hypothesize a role for circrnas in neuronal development. 2 A B C Untreated control Bicucullin-treated No circrna probe Mouse hippocampal slices hybridized with probes for circhomer1_a (green), using ViewRNA Assays, show circrna present both in the cell bodies (DAPI, blue) and dendritic processes (antibody to MAP2, red). circrna is upregulated upon stimulation of neuronal activity (bicucullin), while the linear host transcript showed no change (data not shown). (A) untreated control, (B) bicucullin-treated, (C) no circrna probe. Adapted by permission from Macmillan Publishers Ltd: Nature Neuroscience, advance online publication, 25 February 2015 (doi: /nn.3975). 5

6 WISH RNA in situ hybridization were easier to do? Simple, reproducible, and optimized ViewRNA Assays are designed for either manual or automated sample processing. Optimize your tissue pretreatment conditions once for the tissue type then use the same protocol for any probe. Same slide can be visualized under standard brightfield or fluorescent microscopes No radioactivity No need to make your own probes Walk-away automation Ready for high-throughput tissue staining? ViewRNA ez Assays simplify assay automation with Leica Biosystems Advanced Staining Instruments. Seamless integration into current pathology workflows Hands-free sample processing from tissue preparation to cover-slipping Reagents optimized for automation Brain-derived neurotrophic factor RNA (Bdnf, red) localized in the hippocampus in fresh-frozen rat brain tissue, stained using ViewRNA ez Assays on the Leica Biosystems BOND RX. I tried the new ViewRNA ez Assays on our Leica Biosystems BOND RX and found that the sensitivity and specificity of staining were remarkable. These new assays are easy to run and hands-free. This new platform will be our standard for in situ hybridization. Dr. Paul Shughrue, Prothena Corporation 1 Dewax and permeablilize 2 Hybridize target probe 3 Hybridize amplifiers and label probes 4 Apply detection substrate 5 Mount and image 6

7 Branched-DNA signal amplification and detection delivers up to 96,000x fold amplification for ultra-sensitive signal Each target sequence is identified using synthetic probes consisting of as many as 40 oligonucleotides. (A) Each oligonucleotide has two components: a lower region that is complementary to the target RNA and an upper region that provides the binding site for the branched-dna signal amplification structure. (B) The signal amplification structure is then built onto the probes through sequential hybridization of a series of amplifiers and label probes. The chromogen(s) form a precipitate wherever the target RNA molecules are located. Each signal dot corresponds to one target molecule. To quantify target expression, simply count the dots. A bdna-specific sequence Target RNA-specific sequence B Signal amplification structure Protein complex crosslinked to the RNA Target RNA (partially degraded RNA) bdna-specific sequence from the ViewRNA Probe Distribution of somatostatin (Sst, blue) and serotonin receptor (Htr6, red) RNAs in normal mouse brain. 7

8 WISH you could accelerate assay development? Ask Affy. From genome-wide discovery studies to single-target validation, Affymetrix can deliver probes for the same regions, optimized for each type of assay, to ensure continuity throughout assay development. Discovery High-throughput analysis of whole genomes: CNV, LOH, genes, isoforms, ncrna Validation Validate RNA signatures from large numbers of samples: CNV, LOH, genes, ncrna Final validation and routine assays Visualize transcripts and proteins in tissue sections OncoScan FFPE Assay Kit GeneChip Human Transcriptome Array 2.0 QuantiGene Plex assays ViewRNA Assays (manual and automated) ebioscience IHC antibodies Whether you need a new probe, are exploring new ideas with your ViewRNA Assays, or need guidance navigating regulated environments, our team is ready to assist you. Through our Powered by Affymetrix Program, we will collaborate with you to accelerate the development of your molecular pathology discoveries from research through validation and on to practical clinical applications. Fibronectin 1 mrna transcript (FN1, red) in human breast adenocarcinoma. Discovery through development on one platform to minimize time to market Access to our industrial-scale manufacturing facilities Established regulatory compliance Strategic market development partnership References 1 Shahid M., et al. Branched chain in situ hybridization for albumin as a marker of hepatocellular differentiation: evaluation of manual and automated in situ hybridization platforms. American Journal of Surgical Pathology 39(1):25 34 (2015). 2 You X., et al. Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity. Nature Neuroscience (2015). doi: /nn.3975 Affymetrix, Inc. Tel: Affymetrix UK Ltd. Tel: +44-(0) Affymetrix Japan K.K. Tel: +81-(0) ViewRNA Assays Tel: USB Products Tel: usb.affymetrix.com Please visit our website for international distributor contact information. For Research Use Only: Not for use in diagnostic procedures. Analyte Specific Reagent: Analytical and performance characteristics are not established. General Purpose Reagent: For laboratory use only. P/N GCL05336 Rev Affymetrix, Inc. All rights reserved. Affymetrix, Axiom, Command Console, CytoScan, DMET, GeneAtlas, GeneChip, GeneChip-compatible, GeneTitan, Genotyping Console, mydesign, NetAffx, OncoScan, Powered by Affymetrix, PrimeView, Procarta, QuantiGene, and ViewRNA are trademarks or registered trademarks of Affymetrix, Inc. All other trademarks are the property of their respective owners.