In situ hybridization
A highly valuable tool for providing complete information about location of the gene of interest in the tissue
We use radioactively labeled RNA probes to determine the precise location of low abundance genes in various tissues and for semi-quantitative analysis of mRNA levels in different areas.
If you want to localize your target gene, a fast and often-used approach is immunostaining. This method, however, relies on the availability of specific antibodies, which often are not as specific as they need to be. A particular problem which is often ignored by the commercial suppliers of antibodies is the fact that antibodies can sometimes bind to something that is not your gene of interest. You will get staining, and you think it’s the gene, but it may not be. Radioactive in situ hybridization is incredibly specific, and only binds to the gene you want, giving you an accurate location of the gene of interest. As an example, we often use this method to validate our antibodies.
Although it has been replaced by qPCR in recent years, in situ hybridization is still a highly valuable tool for providing complete information about where the gene of interest is located in the tissue. In comparison to qPCR, it provides you with an estimate of total instead of relative expression. Moreover, it gives you comprehensive details on a cellular level, information that is critical to know how and where your compound impacts.