Abstract
The functional characterization of miRNAs often involves understanding of their spatiotemporal expression, which mostly relies on reporter-based or in situ hybridization studies. The available in situ localization methods follow separate protocols for pre-hybridization, hybridization, post-hybridization, and detection steps for both miRNA and mRNA transcripts in plants. In this study, we present a single method which can be used for whole mount in situ localization of both miRNAs and mRNAs in different plant tissues. Our modified method provides enhanced sensitivity for the localization of miRNA and their target transcripts. Consequently, a less laborious, time-saving, economic and efficient method has been proposed by the modification of pre-hybridization, hybridization, post-hybridization and detection steps.
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References
Aida M, Beis D, Heidstra R, Willemsen V, Blilou I, Galinha C, Nussaume L, Noh Y-S, Amasino R, Scheres B (2004) The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche. Cell 119(1):109–120
Begheldo M, Ditengou FA, Cimoli G, Trevisan S, Quaggiotti S, Nonis A, Palme K, Ruperti B (2013) Whole-mount in situ detection of microRNAs on Arabidopsis tissues using Zip Nucleic Acid probes. Anal Biochem 434(1):60–66. https://doi.org/10.1016/j.ab.2012.10.039
Bleckmann A, Dresselhaus T (2016) Fluorescent whole-mount RNA in situ hybridization (F-WISH) in plant germ cells and the fertilized ovule. Methods 98:66–73. https://doi.org/10.1016/j.ymeth.2015.10.019
Chen X (2012) Small RNAs in development—insights from plants. Curr Opin Genet Dev 22(4):361–367
Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6(3):325–330
de Engler JA, Montagu MV, Engler G (1998) Whole-Mount in situ hybridization in plants. In: Martinez-Zapater JM, Salinas J (eds) Arabidopsis protocols. Humana Press, Totowa, pp 373–384. https://doi.org/10.1385/0-89603-391-0:373
Endrizzi K, Moussian B, Haecker A, Levin JZ, Laux T (1996) The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE. Plant J 10(6):967–979
Friml J, Benkova E, Mayer U, Palme K, Muster G (2003) Automated whole mount localisation techniques for plant seedlings. Plant J 34(1):115–124
Ghosh Dastidar M, Mosiolek M, Bleckmann A, Dresselhaus T, Nodine MD, Maizel A (2016) Sensitive whole mount in situ localization of small RNAs in plants. Plant J 88(4):694–702. https://doi.org/10.1111/tpj.13270
Grunweller A, Hartmann RK (2007) Locked nucleic acid oligonucleotides: the next generation of antisense agents? Biodrugs 21(4):235–243
Hejatko J, Blilou I, Brewer PB, Friml J, Scheres B, Benkova E (2006) In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples. Nat Protoc 1(4):1939–1946. https://doi.org/10.1038/nprot.2006.333
Hernandez-Castellano S, Nic-Can GI, De-la-Pena C (2017) Localization of miRNAs by in situ hybridization in plants using conventional oligonucleotide probes. Methods Mol Biol 1456:51–62. https://doi.org/10.1007/978-1-4899-7708-3_4
Javelle M, Timmermans MC (2012) In situ localization of small RNAs in plants by using LNA probes. Nat Protoc 7(3):533–541. https://doi.org/10.1038/nprot.2012.006
Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3(1):27–29. https://doi.org/10.1038/nmeth843
Lou G, Ma N, Xu Y, Jiang L, Yang J, Wang C, Jiao Y, Gao X (2015) Differential distribution of U6 (RNU6-1) expression in human carcinoma tissues demonstrates the requirement for caution in the internal control gene selection for microRNA quantification. Int J Mol Med 36(5):1400–1408. https://doi.org/10.3892/ijmm.2015.2338
Singh A, Singh S, Panigrahi KC, Reski R, Sarkar AK (2014) Balanced activity of microRNA166/165 and its target transcripts from the class III homeodomain-leucine zipper family regulates root growth in Arabidopsis thaliana. Plant Cell Rep 33(6):945–953. https://doi.org/10.1007/s00299-014-1573-z
Singh A, Gautam V, Singh S, Sarkar Das S, Verma S, Mishra V, Mukherjee S, Sarkar AK (2018) Plant small RNAs: advancement in the understanding of biogenesis and role in plant development. Planta 248(3):545–558. https://doi.org/10.1007/s00425-018-2927-5
Traas J (2008) Whole-mount in situ hybridization of RNA probes to plant tissues. CSH Protoc 2008:pdb.prot4944. https://doi.org/10.1101/pdb.prot4944
Vester B, Wengel J (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43(42):13233–13241. https://doi.org/10.1021/bi0485732
Acknowledgements
V.G., Ar.S., S.C. and S.S. thank Council for Scientific and Industrial Research (CSIR) and Department of Biotechnology, Ministry of Science and Technology (DBT project# BT/PR12766/BPA/118/63/2015), India, and National Institute of Plant Genome Research (NIPGR), New Delhi, India, for funding and internal Grants. S.V. thanks Department of Science and Technology (DST)-Science and Engineering Research Board (SERB) National-PostDoctoral Fellowship (N-PDF grant no. PDF/2016/002423). A.S. thanks NIPGR, New Delhi, India, for fellowship and grants.
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AS, VG, and AS conceived, designed, and performed all of the experiments and related data analysis. SS made the probe constructs. VG, AS, and SC wrote the paper. SV helped in the imaging of the slides. The study was supervised by AS, who provided scientific insight and guidance.
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Gautam, V., Singh, A., Verma, S. et al. Whole mount in situ localization of miRNAs and target mRNA transcripts in plants. 3 Biotech 9, 193 (2019). https://doi.org/10.1007/s13205-019-1704-x
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DOI: https://doi.org/10.1007/s13205-019-1704-x