siRNA-mediated RNA interference in embryonic kidney organ culture.

In principle, treatment of embryonic kidneys growing in organ culture with short interfering RNA (siRNA) offers a powerful means of investigating molecular function quickly and cheaply. Experiments using this approach have yielded significant new data, but they have also highlighted important limitations. Here, we briefly describe the published successes and limitations and present detailed instructions for two methods of siRNA treatment. The first method applies siRNA to intact cultured kidneys; this method is the quicker and easier of the two, but it is the one most affected by problems of siRNA uptake by certain renal tissues. The second method reduces kidney rudiments to a suspension of single cells, applies siRNA at that stage, when the cells are highly accessible, and then reaggregates the kidney; this method is more time-consuming but suffers less from problems of limited uptake. As well as proving instructions for the methods, we provide a brief discussion of necessary controls.