sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants.

Localization of mRNA and small RNAs (sRNA) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study sRNA localization and expression. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol for sRNA-FISH, for efficient fluorescent detection of sRNAs in plants. sRNA-FISH is suitable for application in diverse plant species and tissue types. The use of Locked Nucleic Acid (LNA) probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we successfully detected co-localization of miR2275 and a 24-nt phasiRNA (phased siRNA) in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric in situs, sRNA-FISH signal can be imaged using super-resolution microscopy to examine the sub-cellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy (SR-SIM) and direct stochastic optical reconstruction microscopy (dSTORM). In this study, we describe how we overcame the challenges of adapting fluorescent in situ hybridization for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level. This article is protected by copyright. All rights reserved.