In situ localization of small RNAs in plants by using LNA probes.

Nature Protocols
Marie Javelle, Marja C P Timmermans

Abstract

Small RNAs have crucial roles in numerous aspects of plant biology. Despite our current understanding of their biogenesis and mechanisms of action, the biological function of small RNAs, particularly miRNAs, remains largely unknown. To decipher small RNA function, knowledge about their spatiotemporal patterns of expression is essential. Here we report an in situ hybridization method for the precise localization of small RNAs in plants by using locked nucleic acid (LNA) oligonucleotide probes. This method has been adapted from protocols used to detect messenger RNAs in formaldehyde-fixed and paraffin-embedded tissue sections, but it includes essential optimizations in key prehybridization, hybridization and posthybridization steps. Most importantly, optimization of probe concentration and hybridization temperature is required for each unique LNA probe. We present the detailed protocol starting from sectioned tissues, and we include troubleshooting tips and recommended controls. This method has been used successfully in several plant species and can be completed within 2-6 d.

References

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Citations

Oct 27, 2015·International Journal of Molecular Sciences·Yee-Shan KuHon-Ming Lam
Feb 5, 2016·BMC Plant Biology·Edna Gicela Ortiz MoreaFabio Tebaldi Siveira Nogueira
Feb 26, 2015·Proceedings of the National Academy of Sciences of the United States of America·Jixian ZhaiBlake C Meyers
Feb 27, 2016·Life Sciences·Eng-Soon KhorPooi-Fong Wong
Mar 4, 2014·The Plant Journal : for Cell and Molecular Biology·Geraldo Felipe Ferreira e SilvaFabio Tebaldi Silveira Nogueira
Jul 28, 2016·Frontiers in Plant Science·Fernando E Díaz-ManzanoCarolina Escobar
Nov 1, 2016·Reproduction : the Official Journal of the Society for the Study of Fertility·Chulin YuSheng Cui
Jan 25, 2018·Journal of Experimental Botany·Yanling WangTianlai Li
Dec 24, 2018·The Plant Journal : for Cell and Molecular Biology·Kun HuangJeffrey L Caplan
Jul 18, 2017·The Plant Journal : for Cell and Molecular Biology·Eder Marques da SilvaFabio Tebaldi Silveira Nogueira
Jul 28, 2020·Nucleic Acids Research·Kun HuangJeffrey L Caplan
Feb 9, 2019·Nature Communications·Rui XiaBlake C Meyers
Aug 28, 2014·Beilstein Journal of Nanotechnology·Marcelo RodriguesPeter Ladurner
Mar 27, 2020·Frontiers in Cell and Developmental Biology·Madalina Gabriela BarbuSilviu Cristian Voinea
Aug 8, 2018·Nature Communications·Damianos S SkopelitisMarja C P Timmermans
Dec 16, 2020·Proceedings of the National Academy of Sciences of the United States of America·James W SatterleeMichael J Scanlon
Nov 7, 2017·Developmental Cell·Damianos S SkopelitisMarja C P Timmermans
Jul 13, 2021·Trends in Biochemical Sciences·Raman van WeeChirlmin Joo

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