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The nervous system, especially the brain, is one of the most complex tissues composed of numerous cell types and subtypes organized with delicate topological characteristics, presenting unique challenges to traditional gene expression analysis techniques. The RNAscope™ in situ hybridization assay uniquely addresses these challenges by enabling highly sensitive and specific gene expression analysis at the single-transcript detection level and single-cell resolution, while preserving spatial and morphological context. In particular, the multiplexing capability for the simultaneous detection of multiple neuronal cell markers enables robust gene expression analysis and visualization of distinct cell populations within the nervous system (see Image Gallery).

Furthermore, our BaseScope™ assay goes beyond gene expression detection by allowing for the detection of specific exon-exon junctions in alternatively spliced transcripts in the tissue environment. Alternative splicing is especially prominent in the brain, generating much of the complexity of the brain transcriptome.

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ACD Bio Tile eBook
Learn about the principles of the RNAscope technology, its applications in Neuroscience and how to get the most of your RNAscope experiments in the nervous system.

Spatial RNA Profiling in the Nervous System with the RNAscope Technology

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Neuroscience Research Applications

Channelopathies are responsible for a range of brain disorders and are caused by abnormal ion channel function. There is a need for better understanding of the underlying pathophysiology of these channel-based disorders, as individuals presenting with clinical phenotypes are difficult to diagnose and treat.

Smith et al.,* discovered an unexpected association between one particular channelopathy, connecting sodium channels to cortical folding and brain development. They described an abnormal developmental disorder of the brain, polymicrogyria (PMG), that is associated with pathogenic variants in the sodium channel gene SCN3A. They showed that SCN3A is robustly expressed in cerebral cortex during fetal gestation but downregulated after birth. Conversely, SCN1A is lower during gestation and upregulated postnatally.

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They used the RNAscope Multiplex Fluorescent Assay v2 in fetal human brain and revealed the highest SCN3A expression in the cortical plate (CP), which contains immature neurons, whereas the adult human showed very low SCN3A expression across all cortical layers.

In a Spotlight Interview Dr. Smith explains how the RNAscope ISH assay, with its specificity and multiplex capabilities proved a critical assay for discovering novel findings that provide an important diagnostic marker and challenge the accepted dogma of human cerebral cortical folding.

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Detection, characterization and (co-) localization of neuronal cell types and subcellular target localization.

The RNAscope assay can be used for the visualization of multiple target co-expression patterns or the co-expression of the target(s) of interest with desired cell type markers that characterize particular types of neurons or glia. Commonly used cell type markers to distinguish neuronal and glial target expression patterns include Rbfox3, Aif1, and Gfap for the detection of neurons, microglia and astrocytes, respectively. The striatum harbors two distinct neuronal populations that either express Dopamine Receptor D1 (Drd1) – the striatonigral pathway – or Dopamine Receptor D2 (Drd2) - the striatopallidal pathway. These different neuronal cell types can be accurately detected and visualized using the RNAscope technology.

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Cellular localization of RNA expression in central and peripheral nervous system using RNAscope Technology.

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Visualization of neuronal network activity and plasticity

During learning and memory formation, gene expression is dynamically regulated in response to experience-dependent neuronal activity. In particular, the expression of immediate-early genes (IEGs) including c-fos, and Arc is rapidly and transiently altered in specific neurons or neuronal ensembles in brain areas involved in the learning and memory processing of a certain task/activity. Therefore, the visualization of IEG mRNA expression patterns have widely been used as a molecular readout for neuronal populations that are engaged in generating and encoding long-term memories.

To further characterize activated neuronal cell populations, RNAscope multiplexing capability enables IEG mRNA detection in combination with other targets or cell type markers.

Here, we detect neuronal activity in the mouse brain hippocampus by using the RNAscope assay to visualize Cfos which is a proto-oncogene and transcription factor that is induced within 15 minutes of stimulation (Figure 1). Simultaneously, we used a second probe to detect Chrm3-positive neurons.

Figure 1 Image

Figure 1: Detection of the transcription factor immediate early gene and activity marker Cfos (Green) in combination with the G protein-coupled receptor Cholinergic Receptor Muscarinic 3 (Chrm3, Red) in normal mouse brain hippocampus using the RNAscope™ Multiplex Fluorescent assay on Fresh Frozen tissue samples. Cells are counterstained with DAPI. CA = Cornu Ammonis.

Guzowski JF. Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus 2002; 12:86-104

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Detection of RNA in the central and peripheral nervous system using the RNAscope in situ hybridization assay

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Detection of G protein-coupled receptors (GPCRs)

Detection of mRNA in the nervous system when no (reliable) antibodies are available

IHC is a well-established method for a broad range of applications from discovery to diagnostic and prognostic testing. However, raising antibodies to G-protein coupled receptors (GPCRs) can be challenging due to difficulties obtaining suitable antigen accessibility amongst other reasons as GPCRs are transmembrane proteins that can be expressed at low levels and tend to be unstable when purified.. Ion channels, another class of membrane proteins, also constitute a challenging class of targets for antibody discovery since they must remain membrane-associated to maintain their native conformation. The RNAscope technology is an ideal method to visualize these targets within their morphological context in the central and peripheral nervous systems.

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Detection of G-protein-coupled receptors (GPCRs) in the nervous system using the RNAscope in situ hybridization assay

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References: Hutchings C, Koglin M, Marshall F. Therapeutic antibodies directed at G protein-coupled receptors. mAbs 2010; 2(6), 594-606.

Introducing the highly specific and sensitive miRNAscope™ Assay to detect small RNAs with spatial and morphological context at single-cell resolution.  Leveraging ACD’s RNAscope™ core technology, the miRNAscope assay is designed to enable applications for small RNAs including the detection of antisense oligonucleotides (ASOs), microRNAs (miRNAs), small interfering RNAs (siRNAs), and other smaller RNAs that are 17-50 bases.

  • • Detect and identify cellular subtypes
  • • Visualize gene regulation with morphological context
  • • Validate miRNA biomarkers in intact tissues
  • • Assess small RNA therapeutic delivery mechanism
  • • Evaluate biodistribution and efficacy of therapy
  • • Add a visual dimension to heterogeneous tissue biology and analysis

ACD Bio Image Table

miRNAscope™ assay used to detect miR-214-3p in different species samples including the mouse brain, rat brain, human brain, and mouse liver. miR-214-3p was successfully detected in the mouse, rat, and human brain tissue samples and not in the negative control mouse liver tissue sample.

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Introducing the novel miRNAscope™ in situ hybridization assay for the robust detection of microRNAs and other small RNA targets with spatial resolution

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