Exploring GPCR expression in the eye: From discovery with NGS to spatial and quantitative expression confirmation with RNAscope™ technology

Researcher at the University of Miami, Dr Alexey Pronin, is using ACD’s RNAscope Assays for in situ hybridization (ISH) to detect and localize expression of olfactory signaling genes within the corneal layers of the eye, paving the way for future development of therapeutic targets for ocular diseases.

Can you tell us about your research?

The main goal of our research is to better understand eye physiology, particularly how the eye interacts with the environment and protects itself from potentially harmful agents such as microbes and irritants. More specifically, we are interested in the role of G protein-coupled receptors (GPCRs) in this process, a well-known protein family for therapeutic targets. One of the challenges of working with GPCRs is that they are typically expressed at very low levels, and as proteins they are almost impossible to detect in native tissues. Highly sensitive RNA ISH techniques, such as RNAscope technology, therefore help us in detecting GPCR expression.

How important is spatial information on GPCR expression and why?

Identifying specific cells expressing a particular gene is very important for us. Since many genes that we study have no identified function, such information can provide us with a clue to their function within the eye.

Which techniques do you currently use for your research?

We started our search for novel genes expressed in the eye by performing Next Generation Sequencing (HiSeq) to compile a transcriptome – a library of all mRNAs expressed in the cornea. This provided us with the initial list of GPCRs for further investigation, and we confirmed expression of several interesting genes by RT-PCR. However, neither technique could tell us which exact cells expressed those genes. Other common techniques, such as immunohistochemistry (IHC) and western blot, cannot be used due to lack of appropriate antibodies and low levels of protein expression for GPCRs, and we therefore decided to look towards RISH.

How did you choose which RNA in situ hybridization strategy to use?

Our lab had no prior experience with RNA ISH, and I was looking for an assay that was sufficiently sensitive for GPCR expression, with low background and yet easy to perform. The description of RNAscope technology ticked all the right boxes, so we decided to try it. Like any kit it helps to read the manual and watch videos available online before you run the assay for the first time, and it worked for me on the first try.

Can you tell us how you have applied RNA in situ hybridization within your research, and how do you feel it complements different techniques?

In addition to confirming results obtained through other techniques, such as RT-PCR, RNAscope technology allows us to determine specific cells within a tissue/organ that express our gene of interest. The first RNAscope assays I ran were to confirm expression of several olfactory receptors we identified in the mouse cornea transcriptome, specifically, Olfr558. Interestingly, when I looked at the other parts of the eye, I found Olfr558 mRNA not only where I expected it (in the cornea) but also in the blood vessels of the choroid. To determine the type of vessels expressing Olfr558, I then combined RNAscope ISH with immunohistochemistry, staining the same slides with the antibody against alpha-smooth muscle actin (a marker for ocular arteries). The experiment worked well, revealing co-expression of both molecules and confirming an arterial localization.

In what way has RNAscope technology facilitated your studies?

We’ve discovered a number of advantages of this technique in our research, including:

• The ability to see whether the gene of interest is expressed in a tissue or not
• Spatial information about gene expression tells us which specific cell types express this gene
• The ability for multiplexing means we can now look at two or three genes at the same time
• Providing quantitative information, the assay yields information on relative abundance of the target mRNA
• Relative simplicity and speed means if you have samples and probes ready, you can have an answer in one day

You mention multiplexing. Can you explain further how this benefits your work?

The main advantage of multiplexing is that you can see whether two different genes are expressed in the same cell/tissue or not. Because RNAscope technology can detect expression of multiple genes in parallel, it can tell us whether those genes discovered through RT-PCR are co-expressed in the same cell or not – information that is very valuable and hard to obtain by other means. For example, using multiplexing we showed that both olfactory marker protein and olfactory G protein are expressed in the epithelium of the cornea, yet expression patterns differ. This suggests that these two genes are likely involved in different processes in the eye. Another advantage of multiplexing is simply economical – you save money on kits and tissue slides by looking at two or more genes at the same time.

In conclusion, what do you see for the future of RNA ISH in your research?

We plan to continue using RNAscope technology in our research. There are many more genes in the eye and other parts of the body we would like to look at and see their patterns of expression. One nice feature of RNAscope technology is its ability to generate quantitative information. It not only tells you whether the gene is expressed or not, but also shows relative abundance of mRNA. In the future we would like to take advantage of it and see if expression of the gene of interest changes depending on treatment conditions. After running many assays I would highly recommend the system to other researchers looking to use RNA ISH for their studies.