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Routine, Automated RNA in situ Hybridization Profiling of Target Candidates and Safety Markers in Preclinical Animal Model Tissue Panels using RNAscope® LS Assay on Leica Biosystems’ BOND RX
Author(s) -
Fang Li,
He MingXiao,
Kim Daniel,
Franks Tania,
Roy Marc,
Bunker Chris,
Luo Yuling,
Ma XiaoJun,
Park Emily
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb470
Subject(s) - in situ hybridization , biology , proliferation marker , microbiology and biotechnology , pathology , immunohistochemistry , gene expression , gene , medicine , genetics , immunology
Determining tissue expression profiles of therapeutic target candidates is essential for safety assessment and the potential for adverse events. Immunohistochemistry is often not possible due to lack of reagents with the required specificity and sensitivity coupled with extensive time in attempts to validate IHC. RNAscope® in situ hybridization (ISH) technology is a universal assay able to detect and characterize tissue distribution of any target mRNA. RNAscope ISH has molecular sensitivity, high specificity and robust performance in FFPE tissue. Fully automated RNAscope ISH is performed on Ventana Discovery and Leica Biosystem Bond RX instrumentation; detection of marker RNAs is achieved in 8 hours and visualized via bright field microscopy. Marker RNAs can be quantified with cellular resolution using digital image analysis software. We present RNAscope methods for routine detection of very low copy RNAs, typical of most targets in normal tissues, as well as applications for detection of cell‐type, proliferation and apoptosis markers in animal tissues used in safety assessment and toxicity studies. We demonstrate robust performance in 25 tissues from rat, dog and cynomolgus monkey using probes to low‐, medium‐ and high‐expression housekeeping gene RNAs (POLR2A, PPIB and UBC). Specific signals and well‐maintained morphology was achieved efficiently for all 25 tissues. Based on optimized epitope retrieval conditions, tissues were grouped into two multi‐tissue microarrays for high‐throughput marker analysis. We examined and present tissue‐specific expression patterns of endothelial marker CD31, macrophage marker CD68, proliferation markers Ki‐67 and Cyclin E1, and apoptosis molecules, Puma, Fas/CD95 and DR5. The presentation demonstrates RNAscope as a method that can be replicated in any lab and suitable for efficient and reliable detection of any target candidate or safety/toxicity biomarker mRNA from any species for interrogation in any tissue.