How RNA ISH and IHC can complement each other

To fully understand molecular and cellular mechanisms, researchers often use a variety of technologies. RNA ISH and IHC allow analysis of RNA and Protein, respectively, while also preserving the cellular relationship and tissue architecture. Researchers have found that combining or multiplexing both technologies provides unique information as described in the example below. 

Identify a secreted protein and its origin

As secreted proteins (like growth factors or cytokines) are found outside of the producing cell, IHC will provide the localization of the protein in the tissue matrix but will not provide information for the cell that secreted the protein. To overcome this issue, ISH is used to localize the mRNA precursor of the targeted protein within the secreting cell. Combination of ISH and IHC provides the unique opportunity to identify both the original cell and also localize the protein in the tissue sample. 


Localization of complement factor H gene expression and protein distribution in mouse outer retina. Smit-McBride, Z. et al. 2015. Mol Vis. 21, 110-123 

Identify mRNA synthesis in a complex organ structure

Analysis of RNA expression in a complex organ composed by different but similar types of tissue, may require identification of a specific tissue type to understand origin of the RNA target expression. In such situation, IHC will be used to identify a tissue type based on its membrane protein, while RNAscope ISH will be used for detection and quantification of the RNA target inside this specific tissue. An example of such experiment is described in the article below in which IHC is used to identify 2 tissues in the cornea vessel structure (Endothelial and smooth muscle cells) while RNAscope ISH shows expression of Olf558 only in the smooth muscle cells. 


Expression of olfactory signaling genes in the eye. Pronin, A. 2014. Plos One 9, e96435

Identify gene regulation mechanism

Regulation of gene expression includes a wide range of mechanisms from transcriptional initiation, to RNA processing and to the protein post-translation modification. Simultaneous analysis of RNA and protein expression of the same target genes allow for the identification of potential gene expression regulation mechanisms. For example Polinksy et al. identified that rAAV2/5-mediated GFP protein levels (viral vector used for gene therapy) are decreased in the aged rat striatum, regardless of rat strain and duration of expression. Through combining ISH and IHC they were able to show that RNA expression was at a normal level while the protein expression was decreased, revealing a potential regulation of protein translation rather than any issue with virus transduction in aged rat.


Pentraxins coordinate excitatory synapse maturation and circuit integration of parvalbumin interneurons. Pelkey, K. A. et al. 2015.  Neuron 85, 1257-1272

Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain. Polinski, N. K. et al. 2015. Neurobiol Aging. 36, 1110-1120 

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