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Robust Detection of RNA Biomarkers for Drug Safety Assessment in Preclinical Animal Models by Fully Automated Rnascope® 2.5 LS Assay

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Robust Detection of RNA Biomarkers for Drug Safety Assessment in Preclinical Animal Models by Fully Automated Rnascope® 2.5 LS Assay Application Note Drug Safety Assessment Robust detection of RNA biomarkers for drug safety assessment in preclinical animal models by fully automated RNAscope® 2.5 LS Assay Ming-Xiao He1, Bingqing Zhang1, Daniel Kim1, Tania Franks2, Marc Roy2, Chris Bunker1, Yuling Luo1, Xiao-Jun Ma1, Emily Park1 Preclinical drug safety Abstract eosin (H&E) stain, and also for evaluation of assessment in animal specific biomarkers by immunohistochemistry models is used to evaluate Robust assays to evaluate biomarkers in tissue (IHC). IHC assay has been commonly applied to the pathological effects are needed for preclinical safety assessment and assess therapeutic targets and toxicity-related induced by novel therapeutic toxicity studies. Here we present the application biomarkers. However, consistent and systematic ® agents. Here we present the of the fully automated RNAscope 2.5 LS Assay application of IHC techniques has been hindered use of the RNAscope® 2.5 on Leica BOND RX for RNA in situ hybridization in by inconsistent performance of various antibody LS Assay for the evaluation formalin-fixed paraffin-embedded (FFPE) tissues clones, time-consuming antibody development of biomarkers in tissues from three commonly used animal models (rat, and validation, and general lack of reagents for from three preclinical animal cynomolgus monkey, and dog). We demonstrate some animal models. RNA in situ hybridization models. In this study we: robust assay performance with high signal-to- (ISH) technology presents an attractive alternative noise ratio and well-maintained morphology in 25 method for pathological evaluation of biomarkers • Identify optimal pretreatment different tissues from each species. Based on conditions for different tissues in tissues from various preclinical animal models, these tests, we provide recommendations for in different species because nucleic acid-based probes specific for proper control gene for sample qualification of any biomarkers associated with drug toxicity • Provide recommendations each tissue type, as well as optimal pretreatment or mechanism can be developed and for control gene selection for protocol selection. For specific target RNA validated rapidly. tissue qualification markers, we successfully detected cell type ® specific markers such as CD31 (PECAM1) and The RNAscope technology, an advanced • Detect specific RNA markers CD68, proliferation marker Ki-67 (MKI67), and cell platform for in situ RNA detection, enables in various FFPE tissues from cycle marker Cyclin E1 (CCNE1), as well as detection of almost any RNA biomarker with multiple species apoptosis-related molecules Puma (BBC3), Fas single-molecule detection sensitivity and high (CD95), and DR5 (TNFRSF10B). This study specificity in formalin-fixed paraffin-embedded 2 demonstrates that the RNAscope® 2.5 LS Assay (FFPE) tissues . It provides a universal solution can be an attractive platform for biomarker to characterize tissue distribution of drug targets analysis in tissues for preclinical safety and biomarkers in a highly specific and sensitive assessment and general animal studies. manner, without the need to wait for antibody development and validation. The RNAscope® assay can be performed in fully automated Introduction staining systems, including Ventana Discovery Preclinical drug safety assessment in animal XT, Ventana Discovery ULTRA, and Leica BOND models has been well established as a routine RX instruments. The assay allows visualization of laboratory practice to evaluate the pathological each individual RNA molecule as a punctate dot alterations induced by novel therapeutic agents1. under a standard bright field microscope. The RNA This preliminary evaluation serves a major role dots can be quantified by counting the number of in the development of new treatments prior to signal dots in individual cells, either manually or trials in humans. Histopathological techniques by image analysis tools, including HALOTM (Indica have been traditionally applied for general Labs) and SpotStudioTM (ACD) software. morphological evaluation by hematoxylin and In this study, we demonstrate the feasibility 1 Advanced Cell Diagnostics, Inc 3960 Point Eden Way Hayward, CA, USA 94545 2Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT 06340, USA 1 of evaluating RNA biomarkers in 25 types of tissues from three Automated RNAscope® 2.5 LS assay commonly used animal models using the RNAscope® 2.5 LS Reagent ® Kit-BROWN on the Leica BOND RX instrument. Robust RNA detection Ready-to-use reagents from RNAscope 2.5 LS Reagent Kit-BROWN was achieved following a standard protocol in almost all of the were loaded onto the Leica BOND RX instrument according to the tissues tested, with minor alterations to the prestaining conditions user manual (Doc. No. 322100-USM). FFPE tissue sections were for a few tissues. Here, we identified the threshold of pretreatment baked and deparaffinized on the instrument, followed by epitope needed for different tissue types. We also provide recommendations retrieval (using Leica Epitope Retrieval Buffer 2 at 95°C or at 88°C for for control probes to be applied for tissue qualification, and present 15 min) and protease treatment (15 min at 40°C). Probe hybridization, the evaluation of RNA biomarkers including cell type specific markers signal amplification, colorimetric detection, and counterstaining ® (CD68 and endothelial marker PECAM1), proliferation marker Ki-67 were subsequently performed. A schematic of the RNAscope 2.5 LS (MKI67), and cell cycle marker Cyclin E1 (CCNE1). Overall, our study Assay workflow on Leica BOND RX is presented in Figure 1A. shows that the fully automated RNAscope® 2.5 LS Assay is capable of detecting a broad range of RNA targets in all major tissue types RNAscope® probes with little to no optimization needed, and thus well suited for the Control probes of low-, medium-, and high-expressing housekeeping histopathological evaluation of biomarkers in the assessment of genes (POLR2A, PPIB, and UBC, respectively) were designed and drug-derived toxicity in various tissues and animal models. tested for tissues from each species (Table 2). Because the sequences of the human probes for housekeeping genes are over Materials and Methods 95% homologous to the respective target mRNA sequences of FFPE tissues cynomolgus monkey, human probes were used to test samples of cynomolgus monkey. The bacterial probe DapB was used as a Multiple tissues from three commonly used animals (rat, dog, and negative control. Probes for the cell type biomarkers, proliferation cynomolgus monkey) were harvested using a standard protocol markers, and apoptosis-related molecules used in this study were at the drug safety research and development laboratory of Pfizer- designed for each species. As summarized in Table 3, species- Groton (Table 1). Tissues were cut into 3 mm thickness then fixed specific target probes were tested for all RNA targets except two in 10% neutral-buffered formalin (NBF) for 24-48 hours. Fixed genes, CD68 and KI67, for which human probes were used to detect tissues were dehydrated in a graded series of ethanol and xylene, cynomolgus monkey genes, due to 90-95% homology between the followed by infiltration of melted paraffin at 56°C in an automated probe sequence and target mRNA sequence. processor. Tissue microarrays (TMAs) were constructed, sectioned at a thickness of 5 µm and mounted on the SuperFrost® Plus slides Image acquisition and data analysis (Fisherbrand Cat # 12-550-15). Images were acquired using a Leica Biosystems Aperio AT2 Digital Pathology Scanner. RNA markers were analyzed based on Animal Models: Rat, Dog, and Cynomolgus the average RNA dot number per cell. RNA quantity was scored based on manual counting described as follows. Staining results Hematopoietic Thymus, Lymph Node, Spleen, Tonsil were categorized into five grades according to the number of dots system visualized under a bright-field microscope. 0: No staining or less than GI tract Esophagus, Stomach, Duodenum, Jejunum, Colon 1 dot to every 10 cells (40X magnification); 1+: 1-3 dots/cell (visible at 20-40X magnification); 2+: 4-10 dots/cell, very few dot clusters Urinary tract Kidney, Urinary bladder (visible at 20-40X magnification); 3+: >10 dots/cell, and less than 10% Reproductive system Epididymis, Prostate, Testis, Ovary positive cells have dot clusters (visible at 20X magnification); and 4+: >10 dots/cell, and more than 10% positive cells have dot clusters Skin/soft tissues Skin, Skeletal muscle (visible at 20X magnification). Endocrine glands/ Liver, Pancreas, Adrenal gland exocrine glands Results Respiratory system Lung, Bronchus Optimal pretreatment condition for Nervous system Spinal cord, Retina different tissues in different species ® Cardiovascular The standard protocol of RNAscope 2.5 LS Reagent Kit is designed Heart system to work for the majority of FFPE tissues. In this study, to achieve optimal detection of RNA molecules in each tissue type, we compared TABLE 1. Tissue types from three commonly used animal models. two different pretreatment conditions, standard and mild, with a modification in the epitope retrieval step (Figure 1A). The standard 2 Application Note B Cynomolgus monkey - Testis A RNAscope 2.5 LS Assay workflow Standard Mild √ Steps Description Deparaffinization Epitope retrieval (ER2 95º C/88º C) Hs-POLR2A Pretreat Protease (40º C) H2 O2 block Target Probe Hybridization Hybridize DapB AMP1 AMP2 Rat - Liver AMP3 Amplify Standard √ Mild AMP4 AMP5 AMP6 Rn-Ppib DAB reaction
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