Development of a rapid RNA extraction procedure from urediniospores of the leaf rust fungus, Puccinia triticina.

Obtaining high quality RNA in good quantities is often a requirement for plant-pathogen interaction studies, so it becomes very essential that a highly efficient method should be deployed to isolate RNA from minute quantities of fungal spores. The methods available to date, either require a high quantity of spores or the use of expensive chemicals. The protocol discussed here for RNA isolation from Puccinia triticina pathotype 77-5 urediniospores utilizes TRI Reagent as extraction buffer that is widely used for RNA isolation from plant tissues. Urediniospores have a tough cell wall as compared to other plant cells. Therefore, the protocol was optimized keeping the primary focus on quickly disrupting cell walls. Two different methods, one using a combination of liquid nitrogen and ultrasonic water-bath and the other method using micro-homogenizer were utilized for crushing the spores in the present study. The developed methods do not utilize mortar and pestle, instead they promote direct crushing of urediniospores in tubes; thereby minimizing sample loss and enhancing quality.