Analyzing small and long RNAs in plant development using non-radioactive in situ hybridization

Abstract

In the past decade, hundreds of non-coding RNAs (small and long RNAs) have been identi fi ed as crucial elements in developmental processes and stress response in plants. Among small RNAs, the microRNAs or miRNAs control levels of speci fi c mRNA by inhibiting translation or reducing the stability of their mRNA targets through integration into different ribonucleoproteins (RNP). Spatio-temporal expression of small and long RNAs, using reporter genes or in situ hybridization, is essential to understand their functions. We are interested in understanding the role of various non-coding RNAs (including miRNAs) in the regulation of root and nodule development in legumes, which are agriculturally important crops. Here, we present the protocol we are currently using for detection of small and long RNA in model legume plants and tissues, like nodules and roots. The probe selection, as well as the fi xation and permeabilization steps allowing to preserve tissues and cell integrity and to increase accessibility to RNA targets, will be specifically discussed. © Springer Science+Business Media New York 2013.