Utilization of RNAscope® ISH to detect gene expression in mouse models
Detection of lncRNAin normal mouse brain using the RNAscope Multiplex Fluorescent assay on Fresh Frozen tissue samples.
Detection in the normal mouse brain using the RNAscope Multiplex Fluorescent assay on Fresh Frozen tissue samples OPRM1 OPRD1 OPRK1
Detection of G protein-coupled receptors in normal mouse brain using the RNAscope Multiplex Fluorescent assay on Fresh Frozen tissue samples Chrm3 and DRD2
Expression of Smad7 in Mouse cervix tissue using RNAscope® 2.5 HD Assay Brown
Expression of Smad7 in Mouse stomach tissue using RNAscope® 2.5 HD Assay Brown
Fig. 13: Three-plex detection of glutaminergic neurons, Vglut1 (Red), Vglut2 (Green), and GABAergic neurons Vgat (white) expression in the FFPE mouse brain with RNAscope® Multiplex Fluorescent Assay v2. Nuclei were labeled with DAPI (Blue).
Expression of Arc in Mouse brain tissue using RNAscope® 2.5 HD Assay Brown
Detection of Grin2C (dark brown) and Pvalb (red) mRNA in mouse brain tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)
Expression of Cd274 in Mouse colon tissue using RNAscope® 2.5 HD Assay Brown
Detection of the Chrm3 and cFos in normal mouse brain using the RNAscope Multiplex Fluorescent assay on Fresh Frozen
Detection of Grin2C (dark brown) and Sst (red) mRNA in mouse brain tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)
Expression of Omp(r) andOlfr73(g) in mouse brain fresh-frozen tissue, RNA in situ hybridization (ISH) using RNAscope® Multiplex Fluorescent Assay
Mouse Intestine FFPE Tissue: Ppib mRNA expression using the RNAscope® LS Reagent Kit‒RED
RNA expression of Sc17a6 (Vglut2)(green), SLc17a7 (Vlgut1) (red) and Slc32a1 (white) in mouse brain fresh frozen tissue using RNAscope LS Multiplex Fluorescent Assay
The mouse has become a key mammalian model system for research due to its genetic and physiological similarities to humans. The RNAscope® and BaseScope™ in situ hybridization (ISH) assays can both be applied as methods to detect mouse gene expression with morphological context in a multitude of mouse models:
- Mouse models of human disease, including cardiovascular disease, diabetes and obesity, colitis, cancer of multiple tissue types, and rare genetic diseases
- Humanized and patient-derived xenograft (PDX) mice that require characterization of species-specific cell types
- Models of embryonic development and stem cell function
- Pre-clinical studies for the in vivo assessment of newly developed drug therapies
- Confirmation of targeted gene deletion in whole-body or conditional knockout (KO), knockdown (KD), or knock-in mice
Accurate and reproducible detection of biomarkers is critical for understanding their role in the normal and diseased states. RNA is an ideal indicator of the dynamic genetic expression changes in a cell, but traditional techniques that analyze RNA in bulk tissues or cell populations, such as RT-PCR, mask the cell-to-cell variations in gene expression and result in the loss of information on the spatial relationship of the analyzed cells. Mapping RNA expression to single cells is achievable with the RNAscope® and BaseScope™ ISH assays, which can detect virtually any gene transcript, either protein-coding or non-coding, from any type of tissue derived from various mouse models and is particularly valuable when specific, reliable antibodies are not available.
How RNAscope® assay has been applied to detect gene expression in mouse models:
- Models of human disease:
FNDC4 is a novel secreted factor that is upregulated during inflammation, such as in irritable bowel disease (IBD). To identify the cellular sources of Fndc4 in the inflamed state, Bosma et al. used RNAscope® ISH to show expression of Fndc4 mRNA in the colonic epithelium and immune cells of the distal colon in mice treated with the IBD-inducing chemical DSS.
FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice
- Detection of species-specific cells in humanized mice:
Allweiss et al. established a mouse model where SCID mice were repopulated with primary human hepatocytes and then infected with Hepatitis E virus (HEV). RNAscope® ISH was used to detect HEV in all human hepatocytes but not in non-infected humanized control mice or murine areas of infected areas.
Human liver chimeric mice as a new model of chronic hepatitis E virus infection and preclinical drug evaluation
- Stem cell function:
Lim et al. used RNAscope® ISH to examine the mouse gene expression pattern of 14 Wnts in the inner and outer bulge cells of the hair follicle during the hair cycle and determined that the outer bulge hair cells produce autocrine Wnt signals to maintain stem cell potency in the growth phase. Overall their results suggest that Wnt/β-Catenin signaling is a constant and persistent feature of the hair follicle stem cell (HFSC) niche.
Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling
- Pre-clinical studies:
Delivery of DNA-based gene therapy has posed challenges, however RNA therapy is an attractive option because it provides transient production of therapeutic proteins without the need for nuclear delivery or risk of insertional mutagenesis. DeRosa et al. used lipid nanoparticles to deliver hEPO mRNA by IV injection to mice and RNAscope® ISH confirmed delivery of hEPO mRNA to hepatocytes. Following mRNA delivery there was increased EPO mRNA, serum protein, and hematocrit, showing that exogenous mRNA-derived protein maintains normal activity.
Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system
- Confirmation of gene deletion in knockout mice:
Perdigoto et al. used RNAscope® ISH to confirm the loss of Sonic hedgehog (Shh) expression in Shh KO mice at embryonic day 16 and postnatal day 0.
Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development