Abstract
Over the years, in situ hybridization has been used for visualizing spatial gene expression patterns. Direct comparison of the expression patterns of different genes is useful for studying various biological activities in many situations. If we can distinguish signals derived from different probes in the same hybridization reaction, the localization of multiple gene transcripts can be detected simultaneously. In this chapter, we describe a two-color in situ hybridization procedure, which enables us to compare the expression patterns of two genes in a single tissue section. First, we explain how to prepare RNA probes and tissue samples. Then, we explain how to perform an in situ experiment, including pre-hybridization, hybridization, post-hybridization, and detection steps. A stepwise detection procedure is used to obtain the two color signals.
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References
Heisler MG, Ohno C, Das P, Sieber P, Reddy GV, Long JA, Meyerowitz EM (2005) Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem. Curr Biol 15:1899–1911. https://doi.org/10.1016/j.cub.2005.09.052
Gordon SP, Heisler MG, Reddy GV, Ohno C, Das P, Meyerowitz EM (2007) Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development 134:3539–3548. https://doi.org/10.1242/dev.010298
Tucker MR, Hinze A, Tucker EJ, Takada S, Jurgens G, Laux T (2008) Vascular signaling mediated by ZWILLE potentiates WUSCHEL function during shoot meristem stem cell development in the Arabidopsis embryo. Development 135:2839–2843. https://doi.org/10.1242/dev.023648
Mcfadden GI (1989) In situ hybridisation in plants - from macroscopic to ultrastructural resolution. Cell Biol Int Rep 13:3–21. https://doi.org/10.1016/S0309-1651(89)80004-9
Toriba T, Suzaki T, Yamaguchi T, Ohmori Y, Tsukaya H, Hirano H-Y (2010) Distinct regulation of adaxial-abaxial polarity in anther patterning in rice. Plant Cell 22:1452–1462. https://doi.org/10.1105/tpc.110.075291
Bradley D, Ratcliffe O, Vincent C, Carpenter R, Coen E (1997) Inflorescence commitment and architecture in Arabidopsis. Science 275:80–83. https://doi.org/10.1126/science.275.5296.80
Fobert PR, Coen ES, Murphy GJP, Doonan JH (1994) Patterns of cell-division revealed by transcriptional regulation of genes during the cell-cycle in plants. EMBO J 13:616–624
Souer E, Rebocho AB, Bliek M, Kusters E, de Bruin RAM, Koes R (2008) Patterning of inflorescences and flowers by the F-box protein DOUBLE TOP and the LEAFY homolog ABERRANT LEAF AND FLOWER of petunia. Plant Cell 20:2033–2048. https://doi.org/10.1105/tpc.108.060871
Sato Y, Sentoku N, Nagato Y, Matsuoka M (1998) Isolation and characterization of a rice homeobox gene, OSH15. Plant Mol Biol 38:983–998. https://doi.org/10.1023/A:1006065622251
Tabuchi H, Zhang Y, Hattori S, Omae M, Shimizu-Sato S, Oikawa T, Qian Q, Nishimura M, Kitano H, Xie H, Fang XH, Yoshida H, Kyozuka J, Chen F, Sato Y (2011) LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems. Plant Cell 23:3276–3287. https://doi.org/10.1105/tpc.111.088765
Kouchi H, Sekine M, Hata S (1995) Distinct classes of mitotic cyclins are differentially expressed in the soybean shoot apex during the cell cycle. Plant Cell 7:1143–1155. https://doi.org/10.1105/tpc.7.8.1143
Schenborn ET, Mierendorf RC (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res 13:6223–6236
Kouchi H, Hata S (1993) Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Mol Gen Genet 238:106–119
Weigel D, Glazebrook J (2002) ARABIDOPSIS : a Laboratory Manual. Cold Spring Harbor laboratory press. Cold Spring Harbor, New York
Acknowledgment
We thank Dr. Wakana Tanaka for critical reading of the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (25113008).
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Toriba, T., Hirano, HY. (2018). Two-Color In Situ Hybridization: A Technique for Simultaneous Detection of Transcripts from Different Loci. In: Yamaguchi, N. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 1830. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8657-6_16
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DOI: https://doi.org/10.1007/978-1-4939-8657-6_16
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