Abstract
Among the epigenetic mechanisms studied with a greater interest in the last decade are the microRNAs (miRNAs). These small noncoding RNA sequences that are approximately 17–22 nucleotides in length play an essential role in many biological processes of various organisms, including plants. The analysis of spatiotemporal expression of miRNAs provides a better understanding of the role of these small molecules in plant development, cell differentiation, and other processes; but such analysis is also an important method for the validation of biological functions. In this work, we describe the optimization of an efficient protocol for the spatiotemporal analysis of miRNA by in situ hybridization using different plant tissues embedded in paraffin. Instead of LNA-modified probes that are typically used for this work, we use conventional oligonucleotide probes that yield a high specificity and clean distribution of miRNAs.
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References
Chen X (2005) MicroRNA biogenesis and function in plants. FEBS Lett 579:5923–5931
Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136:642–655
Axtell MJ (2013) Classification and comparison of small RNAs from plants. Ann Rev Plant Biol 64:137–159
Rhoades J, Bartel DP, Bartel B (2006) miRNAs and their regulatory roles in plants. Ann Rev Plant Physiol 57:19–53
Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190
Xie Z, Khanna K, Ruan S (2010) Expression of microRNAs and its regulation in plants. Sem Cell Dev Biol 21:790–797
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Lin Y, Lai Z (2013) Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in Longan (Dimocarpus longan Lour). PLoS One 8, e60337
Liu X, Huang J, Wang Y, Khanna K, Xie Z, Owen HA, Zhao D (2010) The role of floral organs in carpels, an Arabidopsis loss-of-function mutation in MicroRNA160a, in organogenesis and the mechanism regulating its expression. Plant J 62:416–428
Wu XM, Liu M, Ge X, Xu Q, Guo W (2011) Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange. Planta 233:495–505
Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157
Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:154–158
Zhang Z, Yu J, Li D, Liu F, Zhou X, Wang T, Ling Y, Su Z (2010) PMRD: plant microRNA database. Nucleic Acids Res 38:D806–D881
Sun X, Dong B, Yin L, Zhang R, Du W, Liu D, Shi N, Li A, Liang Y, Mao L (2013) PMTED: a plant microRNA target expression database. BMC Bioinformatics 14:174
Nelson PT, Baldwin DA, Scearce LM, Oberholtzer JC, Tobias JW, Mourelatos Z (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1:155–161
Eldem V, Okay S, Ünver T (2013) Plant microRNAs: new players in functional genomics. Turk J Agric For 37:21
Unver T, Namuth-Covert D, Budak H (2009) Review of current methodological approaches for characterizing microRNAs in plants. Inter J Plant Gen 1:1–11
Tran N (2009) Fast and simple micro-RNA northern blots. Biochem Insights 2:1–3
Alastair W, Hye-Jin L, Wark D (2008) Multiplexed detection methods for profiling microRNA expression in biological samples. Angew Chem Int Ed 47:644–652
Kidner C, Timmermans M (2006) In situ hybridization as a tool to study the role of microRNAs in plant development. In: Ying SY (ed) MicroRNA protocols. Humana Press, Totowa, pp 159–179
Song R, Ro S, Yan W (2010) In situ hybridization detection of microRNAs. Methods Mol Biol 628:287–294
Javelle M, Timmermans MC (2012) In situ localization of small RNAs in plants by using LNA probes. Nat Protocols 7:533–544
Nuovo GJ (2008) In situ detection of precursor and mature microRNAs in paraffin embedded, formalin fixed tissues and cell preparations. Methods 44:39–46
Quiroz-Figueroa FR, Monforte-González M, Galaz-Avalos RM, Loyola-Vargas VM (2006) Direct somatic embryogenesis in Coffea canephora. In: Loyola-Vargas VM, Vázquez-Flota FA (eds) Plant cell culture protocols. Humana Press, Totowa, NJ, pp 111–117
Acknowledgement
This work was supported by grants from CONSEJO NACIONAL DE CIENCIA Y TECNOLOGÍA (CONACYT) to C.D. (178149), CONACYT-scholarship to S.H.C. (271240), and Cátedras-CONACYT ICC1 to G.N.C.
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Hernández-Castellano, S., Nic-Can, G.I., De-la-Peña, C. (2017). Localization of miRNAs by In Situ Hybridization in Plants Using Conventional Oligonucleotide Probes. In: Kovalchuk, I. (eds) Plant Epigenetics. Methods in Molecular Biology, vol 1456. Humana Press, Boston, MA. https://doi.org/10.1007/978-1-4899-7708-3_4
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DOI: https://doi.org/10.1007/978-1-4899-7708-3_4
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