Simultaneous Detection of mRNA and Protein

The challenge in trying to detect both protein and mRNA in your embryos is finding the conditions which will yield an acceptable result for each. The mild proteinase digestion of the embryos results in the optimum hybridization of probes to their targets, giving you the best RNA signal, but will destroy many of the protein epitopes that your antibody should recognize, giving you the worst protein signal, or none at all. Here are some methods that get around the ProtK treatment, thus allowing your antibody to work while still obtaining a good signal from your RNA probe.

These methods assume that your antibody will still work well even after its protein targets have been slow-cooked in hybridization solution. For the antibodies I have tried, the hybridization itself does not seem to reduce the quality of antibody staining, but that might not always hold true. So what works best? All three methods have worked for me, but I haven't tried them in parallel so I can't make a fair comparison. They're all worth trying with your particular RNA probe and antibody combination. Parallel experiments with and without ProtK made me conclude that ProtK treatment is still essential for the best RNA signal and you will have to sacrifice some percentage of it in order to see your protein as well. If you have great signal to begin with, you will get your cake and eat it too. But what if the harsh conditions of hybridization do completely destroy the epitopes normally recognized by your antibody? Another approach is to perform a normal antibody staining procedure before hybridization. Subsequent post-fixation of the embryos allows the secondary detection reagents to persist through the hybridization steps, to be visualized later along with the RNA probe. Two variations on this theme are described in Goto and Hayashi (1997) and Sturtevant et al. (1993).

Next: Alternative Methods for Fluorescent Detection

Previous: Presentation of Embryos for Fluorescence Microscopy

Back to Index