Preclinical drug safety assessment in animal models is used to evaluate the pathological effects induced by novel therapeutic agents. Here we present the use of the RNAscope® 2.5 LS Assay for the evaluation of biomarkers in tissues from three preclinical animal models. In this study we:
- Identify optimal pretreatment conditions for different tissues in different species
- Provide recommendations for control gene selection for tissue qualification
- Detect specific RNA markers in various FFPE tissues from multiple species
Abstract
Robust assays to evaluate biomarkers in tissue are needed for preclinical safety assessment and toxicity studies. Here we present the application of the fully automated RNAscope® 2.5 LS Assay on Leica BOND RX for RNA in situ hybridization in formalin-fixed paraffin-embedded (FFPE) tissues from three commonly used animal models (rat, cynomolgus monkey, and dog). We demonstrate robust assay performance with high signal-tonoise ratio and well-maintained morphology in 25 different tissues from each species. Based on these tests, we provide recommendations for proper control gene for sample qualification of each tissue type, as well as optimal pretreatment protocol selection. For specific target RNA markers, we successfully detected cell type specific markers such as CD31 (PECAM1) and CD68, proliferation marker Ki-67 (MKI67), and cell cycle marker Cyclin E1 (CCNE1), as well as apoptosis-related molecules Puma (BBC3), Fas (CD95), and DR5 (TNFRSF10B). This study demonstrates that the RNAscope® 2.5 LS Assay can be an attractive platform for biomarker analysis in tissues for preclinical safety assessment and general animal studies.
Introduction
Preclinical drug safety assessment in animal models has been well established as a routine laboratory practice to evaluate the pathological alterations induced by novel therapeutic agents1. This preliminary evaluation serves a major role in the development of new treatments prior to trials in humans. Histopathological techniques have been traditionally applied for general morphological evaluation by hematoxylin and eosin (H&E) stain, and also for evaluation of specific biomarkers by immunohistochemistry (IHC). IHC assay has been commonly applied to assess therapeutic targets and toxicity-related biomarkers. However, consistent and systematic application of IHC techniques has been hindered by inconsistent performance of various antibody clones, time-consuming antibody development and validation, and general lack of reagents for some animal models. RNA in situ hybridization (ISH) technology presents an attractive alternative method for pathological evaluation of biomarkers in tissues from various preclinical animal models, because nucleic acid-based probes specific for any biomarkers associated with drug toxicity or mechanism can be developed and validated rapidly. The RNAscope® technology, an advanced platform for in situ RNA detection, enables detection of almost any RNA biomarker with single-molecule detection sensitivity and high specificity in formalin-fixed paraffin-embedded (FFPE) tissues2. It provides a universal solution to characterize tissue distribution of drug targets and biomarkers in a highly specific and sensitive manner, without the need to wait for antibody development and validation. The RNAscope® assay can be performed in fully automated staining systems, including Ventana Discovery XT, Ventana Discovery ULTRA, and Leica BOND RX instruments. The assay allows visualization of each individual RNA molecule as a punctate dot under a standard bright field microscope. The RNA dots can be quantified by counting the number of signal dots in individual cells, either manually or by image analysis tools, including HALOTM (Indica Labs) and SpotStudioTM (ACD) software. In this study, we demonstrate the feasibility of evaluating RNA biomarkers in 25 types of tissues from three commonly used animal models using the RNAscope® 2.5 LS Reagent Kit-BROWN on the Leica BOND RX instrument. Robust RNA detection was achieved following a standard protocol in almost all of the tissues tested, with minor alterations to the prestaining conditions for a few tissues. Here, we identified the threshold of pretreatment needed for different tissue types. We also provide recommendations for control probes to be applied for tissue qualification, and present the evaluation of RNA biomarkers including cell type specific markers (CD68 and endothelial marker PECAM1), proliferation marker Ki-67 (MKI67), and cell cycle marker Cyclin E1 (CCNE1). Overall, our study 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 with little to no optimization needed, and thus well suited for the histopathological evaluation of biomarkers in the assessment of drug-derived toxicity in various tissues and animal models.>> Download the full Application Note as PDF
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