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Title: sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants
Summary

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescentin situhybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescentin situdetection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA 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 colorimetricin situhybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.

 
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Award ID(s):
1754097 1822293
NSF-PAR ID:
10460789
Author(s) / Creator(s):
 ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
The Plant Journal
Volume:
98
Issue:
2
ISSN:
0960-7412
Page Range / eLocation ID:
p. 359-369
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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