We want to perform outside out patching in oocytes. Therfore, we injected mRNA in 1:10 (alpha:beta) ratio. Patching was done after 3 days of injection and we applied 50 micromolar of Ach solution, but we failed to get any result. From literature we learned that it is also possible to inject cDNA. Is injecting cDNA is better over mRNA for getting current?
My experience was that cDNA nuclear injections produced bigger expression than mRNA injected into cytoplasm (this was with GABAa subunits). RNA production in vitro can be tricky but with the right precautions it's not an issue. Injecting RNA can be tricky only because it is chemically labile- so have to have RNAase free surfaces and pipettes etc. Remember that despite all precautions some batch of oocytes may not express or have a low expression level for unknown reasons. Overall a bit of persistence and precaution will help you to succeed. Best of luck!
Injecting mRNA is much easier and typically gives more consistent oocyte to oocyte expression. There are some labs that routinely inject cDNA and get it to work, but if look through the literature most labs still use RNA.
A few important considerations:
First, if you inject cDNA make sure you inject directly into the nucleus and keep the volume down (5-10 nL), you can practice nuclear injections using dye (diluted DNA loading dye is a good choice). After you inject the dye, pop the oocytes and see if you hit the target, the nucleus will be right under the dark pole and should be full of blue dye.
Second consideration in the vector you use. A CMV promoter works well for cDNA injected expression.
Finally, have you tested your oocytes for whole cell currents (two-electrode voltage clamp)? It is a lot easier to determine your expression using TEVC than doing outside-out patches. If you do not have large enough currents in whole cell, you will have a hard time getting currents in outside-out patches.
Good luck.
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