How RNA ISH and IHC can complement each other
Understanding molecular mecanisms requires to use a variety of technologies in concernt. Because RNA ISH and IHC allow analysis of RNA and Protein while preserving the cellular relationship and tissues texture, using both technologies provide unique informations as describe in the example below.
Identify a secreted protein and its origin
As secreted protein (like growth factors or cytokines) are found outside of the the producing cell, IHC will provide the localization of the protein in the tissue matrix but will not provide information on the cell secreted the protein. To overcome this issue, ISH will be use to localize the mRNA precursor of the targeted protein in the secreting cell. Combination of ISH and IHC provides the unique possibility to identify the original producing cell and 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 complexe 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. Concommitant analysis of RNA and protein expression of the same target gene allow identification of a potential genhe expression regulation mechanism. 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