RNAscope Assay

RNAscope assay is a novel proprietary technology for RNA in situ hybridization (ISH) that was developed by Advanced Cell Diagnostics, Inc. (ACD Bio). The main difference of RNAscope with standard RNA ISH is the use of a double-Z probe design strategy: target-specific probes (ZZ pairs) minimize nonspecific off-target noise signals. This is turn allows highly specific staining capable of single-molecule detection in cultivated cells or tissues (1).   RNAscope assay is used in a wide range of applications: gene expression, point mutation, antibody, vaccine or other therapeutic agents’ validation, etc…(2,3,4).

RNAscope assay can be used different kind of tissues:

  1. Formalin-fixed paraffin-embedded tissue: due to high auto-fluorescence, paraffin sections requires chromogenic staining.
  2. Fresh-frozen tissues: offers highest reactivity, optimum for tissue with low blood content (brain)
  3. Perfusion fixed frozen tissues: for tissues with high blood content (heart)

Three RNAscope assay kits developed by ACD Bio are widely used in biomedical research:

  1. RNAscope 2.5 HD Assay: Chromogen-based assay that produces sensitive and reliable result. Ideal for projects focusing on expression of one (possibly two) specific genes. The sample will be imaged in transmission mode.
  2. RNAscope Fluorescent Multipex Assay: fluorescence-based assay that allows simultaneous detection of up to 5 genes in complex tissues such as brain and tumors. If imaging on the MIC slide scanner, the sample must be stained with DAPI for tissue detection and focusing, thereby limiting the number of genes to 4 (green, red, far-red and infra-red fluorescence channels).
  3. RNAscope HiPlex Assay: Powerful fluorescence-based assay that allows detection of up to 15 genes in complex tissues (12 markers plus DAPI on the MIC slide scanner).

The MIC is fully equipped to handle RNAscope assays on fresh-frozen or perfusion-fixed frozen tissue. A fully equipped RNAscope bench that includes an ACD Bio HybEZ II Oven is available. Samples that do not use the infra-red (Cy7) channel of the ACD kit can be imaged on any microscope. Sample with the Cy7 channel must be imaged on either the Zeiss Axioscan automated slide scanner or the Zeiss wide-field microscope.

Note the MIC will not process large batches of RNAscope samples but rather train you on how to perform the staining. Contact Dr. Ling Yi (yil@mail.nih.gov) for details.

Refer to caption.
RNAscope Bench in MIC/NICHD

Additional resources

ACD RNAscope Multiplex Fluorescent Reagent Kit V2 User Manual external link (PDF 796 KB)
RNAscope Multiplex V2 Protocol and Checklist for Fresh Frozen Sections (PDF 153 KB)
RNAscope Multiplex V2 Protocol and Checklist for Fixed Frozen Sections (PDF 183 KB)

ACD RNAscope HiPlex12 Reagent Kit V2 User Manual external link (PDF 1 MB)
RNAscope HiPlex12 V2 Protocol and Checklist for Fresh Frozen Sections (PDF 194 KB)
RNAscope HiPlex12 V2 Protocol and Checklist for Fixed Frozen Sections (PDF 223 KB)


  1. Wang F, Flanagan J, Su N, Wang LC, Bui S, et al. RNAscope: a a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 2012 Jan;14(1):22-9.
  2. Corbett KS, Flynn B, Foulds KE, Francica JR, Boyoglu-Barnum S, et al. Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates. N Engl J Med. 2020 Oct 15;383(16):1544-1555.
  3. Yin J, Spillman E, Cheng ES, Short J, Chen Y, et al. Brain-specific lipoprotein receptors interact with astrocyte derived apolipoprotein and mediate neuron-glia lipid shuttling. Nat Commun. 2021 Apr 23;12(1):2408.
  4. Alkaslasi MR, Piccus ZE, Hareendran S, Silberberg H, Chen L, et al.  Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord. Nat Commun. 2021 Apr 30;12(1):2471.
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