Protocols for Small RNA FISH in Plants

doi:10.11983/CBB21057

Abstract

Abstract: Small RNAs are a type of small nucleotide molecules that are essential for plant growth and development, playing a key role in variety of life processes and in response to stresses. Research on the location of small RNAs could discover their functions in plants. Small RNA FISH is a qualitative or semi-quantitative analysis of small RNA in organisms by fluorescence detection technology. At present, this technology has been widely used in animals, but it is still less applied in plants. This article introduces the specific operation procedures and attentions based on ultra-high resolution microscopy that combines locked nucleic acid (LNA) probe in situ hybridization with immunofluorescence. This protocol can be used to detect the expression and localization of small RNA in plant tissues.

Key words: fluorescent in situ hybridization, small RNA, plants

Binbin Hu, Zhihui Xue, Cui Zhang. Protocols for Small RNA FISH in Plants[J]. Chinese Bulletin of Botany, 2021, 56(3): 330-338.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB21057

https://www.chinbullbotany.com/EN/Y2021/V56/I3/330

Figures/Tables 2

References 55

1	Baumberger N, Baulcombe DC (2005). Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 102, 11928-11933. DOI URL
2	Bhogale S, Mahajan AS, Natarajan B, Rajabhoj M, Thulasiram HV, Banerjee AK (2014). MicroRNA156: a potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena. Plant Physiol 164, 1011-1027. DOI URL
3	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. DOI PMID
4	Bruno L, Muto A, Spadafora ND, Iaria D, Chiappetta A, Van Lijsebettens M, Bitonti MB (2011). Multi-probe in situ hybridization to whole mount Arabidopsis seedlings. Int J Dev Biol 55, 197-203. DOI URL
5	Bruno L, Ronchini M, Gagliardi O, Corinti T, Chiappetta A, Gerola P, Bitonti MB (2015). Analysis of ATGUS1 and ATGUS2 in Arabidopsis root apex by a highly sensitive TSA-MISH method. Int J Dev Biol 59, 221-228. DOI URL
6	Buhtz A, Springer F, Chappell L, Baulcombe DC, Kehr J (2008). Identification and characterization of small RNAs from the phloem of Brassica napus. Plant J 53, 739-749. DOI URL
7	Carlsbecker A, Lee JY, Roberts CJ, Dettmer J, Lehesranta S, Zhou J, Lindgren O, Moreno-Risueno MA, Vatén A, Thitamadee S, Campilho A, Sebastian J, Bowman JL, Helariutta Y, Benfey PN (2010). Cell signaling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465, 316-321. DOI PMID
8	Chen XM (2004). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 2022-2025. DOI URL
9	Chen XM (2009). Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25, 21-44. DOI URL
10	Chitwood DH, Nogueira FT, Howell MD, Montogmery TA, Carrington JC, Timmermans MC (2008). Pattern formation in leaves via small RNA mobility. Dev Biol 319, 587-588.
11	Chitwood DH, Nogueira FTS, Howell MD, Montgomery TA, Carrington JC, Timmermans MCP (2009). Pattern formation via small RNA mobility. Gene Dev 23, 549-554. DOI URL
12	Fischer AH, Jacobson KA, Rose J, Zeller R (2008). Paraffin embedding tissue samples for sectioning. CSH Protoc 2008, pdb.prot4989.
13	Himanen K, Woloszynska M, Boccardi TM, De Groeve S, Nelissen H, Bruno L, Vuylsteke M, Van Lijsebettens M (2012). Histone H2b monoubiquitination is required to reach maximal transcript levels of circadian clock genes in Arabidopsis. Plant J 72, 249-260. DOI URL
14	Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, Givan SA, Kasschau KD, Carrington JC (2007). Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidop- sis reveals dependency on miRNA- and tasiRNA-directed targeting. Plant Cell 19, 926-942. PMID
15	Huang K, Baldrich P, Meyers BC, Caplan JL (2019). sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants. Plant J 98, 359-369. DOI
16	Huen AK, Rodriguez-Medina C, Ho AYY, Atkins CA, Smith PMC (2017). Long-distance movement of phospha- te starvation-responsive microRNAs in Arabidopsis. Plant Biol 19, 643-649. DOI URL
17	Javelle M, Timmermans MCP (2012). In situ localization of small RNAs in plants by using LNA probes. Nat Protoc 7, 533-541. DOI PMID
18	Jiang JM (2019). Fluorescence in situ hybridization in plants: recent developments and future applications. Chromoso- me Res 27, 153-165.
19	Jones-Rhoades MW, Bartel DP, Bartel B (2006). Micro- RNAs and their regulatory roles in plants. Annu Rev Plant Biol 57, 19-53. PMID
20	Jung JH, Park CM (2007). MIR166/ 165 genes exhibit dynamic expression patterns in regulating shoot apical meris- tem and floral development in Arabidopsis. Planta 225, 1327-1338. DOI URL
21	Kidner C, Timmermans M (2006). In situ hybridization as a tool to study the role of microRNAs in plant development. Methods Mol Biol 342, 159-179.
22	Kim VN, Han JJ, Siomi MC (2009). Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10, 126-139.
23	Knauer S, Holt AL, Rubio-Somoza I, Tucker EJ, Hinze A, Pisch M, Javelle M, Timmermans MC, Tucker MR, Laux T (2013). A protodermal miR394 signal defines a region of stem cell competence in the Arabidopsis shoot meristem. Dev Cell 24, 125-132. DOI URL
24	Kurihara Y, Watanabe Y (2004). Arabidopsis micro-RNA biogenesis through dicer-like 1 protein functions. Proc Natl Acad Sci USA 101, 12753-12758. DOI URL
25	Lamb JC, Danilova T, Bauer MJ, Meyer JM, Holland JJ, Jensen MD, Birchler JA (2007). Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes. Genetics 175, 1047-1058. DOI URL
26	Lee Y, Kim M, Han JJ, Yeom KH, Lee S, Baek SH, Kim VN (2004). MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23, 4051-4060. DOI URL
27	Levsky JM, Singer RH (2003). Fluorescence in situ hybridization: past, present and future. J Cell Sci 116, 2833-2838. DOI URL
28	Li JJ, Yang ZY, Yu B, Liu J, Chen XM (2005). Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis. Curr Biol 15, 1501-1507. DOI URL
29	Li S, Wang XT, Xu WY, Liu T, Cai CM, Chen LY, Clark CB, Ma JX (2021). Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts. Nat Plants 7, 50-59. DOI URL
30	Lin SI, Chiang SF, Lin WY, Chen JW, Tseng CY, Wu PC, Chiou TJ (2008). Regulatory network of microRNA399 and PHO2 by systemic signaling. Plant Physiol 147, 732-746. DOI URL
31	Liu QL, Yao XZ, Pi LM, Wang H, Cui XF, Huang H (2009). The ARGONAUTE10 gene modulates shoot apical meris- tem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis. Plant J 58, 27-40. DOI URL
32	Lu J, Tsourkas A (2009). Imaging individual microRNAs in single mammalian cells in situ. Nucleic Acids Res 37, e100. DOI URL
33	Moissiard G, Parizotto EA, Himber C, Voinnet O (2007). Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins. RNA 13, 1268-1278. DOI URL
34	Nodine MD, Bartel DP (2010). MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis. Gene Dev 24, 2678-2692. DOI URL
35	Nogueira FTS, Chitwood DH, Madi S, Ohtsu K, Schnable PS, Scanlon MJ, Timmermans MCP (2009). Regulation of small RNA accumulation in the maize shoot apex. PLoS Genet 5, e1000320. DOI URL
36	Nogueira FTS, Madi S, Chitwood DH, Juarez MT, Timmer- mans MCP (2007). Two small regulatory RNAs establish opposing fates of a developmental axis. Gene Dev 21, 750-755. PMID
37	Ori N, Cohen AR, Etzioni A, Brand A, Yanai O, Shleizer S, Menda N, Amsellem Z, Efroni I, Pekker I, Alvarez JP, Blum E, Zamir D, Eshed Y (2007). Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nat Genet 39, 787-791. DOI URL
38	Pagliarani C, Gambino G (2019). Small RNA mobility: spread of RNA silencing effectors and its effect on developmental processes and stress adaptation in plants. Int J Mol Sci 20, 4306. DOI URL
39	Pant BD, Buhtz A, Kehr J, Scheible WR (2008). MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J 53, 731-738. DOI URL
40	Pant BD, Musialak-Lange M, Nuc P, May P, Buhtz A, Kehr J, Walther D, Scheible WR (2009). Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. Plant Physiol 150, 1541-1555. DOI URL
41	Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O (2004). In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Gene Dev 18, 2237-2242. PMID
42	Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005). Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 102, 3691-3696. DOI URL
43	Raman S, Greb T, Peaucelle A, Blein T, Laufs P, Theres K (2008). Interplay of miR164, CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana. Plant J 55, 65-76. DOI URL
44	Rozier F, Mirabet V, Vernoux T, Das P (2014). Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization. Nat Protoc 9, 2464-2475. DOI URL
45	Skopelitis DS, Hill K, Klesen S, Marco CF, von Born P, Chitwood DH, Timmermans MCP (2018). Gating of miRNA movement at defined cell-cell interfaces governs their impact as positional signals. Nat Commun 9, 3107. DOI PMID
46	Tirichine L, Andrey P, Biot E, Maurin Y, Gaudin V (2009). 3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei. Plant Methods 5, 11. DOI PMID
47	Vaucheret H, Vazquez F, Crété P, Bartel DP (2004). The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Gene Dev 18, 1187-1197. PMID
48	Wollmann H, Mica E, Todesco M, Long JA, Weigel D (2010). On reconciling the interactions between APETA- LA2, miR172 and AGAMOUS with the ABC model of flower development. Development 137, 3633-3642. DOI PMID
49	Woloszynska M, Le Gall S, Neyt P, Boccardi TM, Grasser M, Längst G, Aesaert S, Coussens G, Dhondt S, Van De Slijke E, Bruno L, Fung-Uceda J, Mas P, Van Montagu M, Inzé D, Himanen K, De Jaeger G, Grasser KD, Van Lijsebettens M (2019). Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators. Proc Natl Acad Sci USA 116, 8060-8069. DOI URL
50	Xue ZH, Liu LY, Zhang C (2020). Regulation of shoot apical meristem and axillary meristem development in plants. Int J Mol Sci 21, 2917. DOI URL
51	Yang WB, Schuster C, Prunet N, Dong QK, Landrein B, Wightman R, Meyerowitz EM (2020). Visualization of protein coding, long noncoding, and nuclear RNAs by fluorescence in situ hybridization in sections of shoot apical meristems and developing flowers. Plant Physiol 182, 147-158. DOI URL
52	Yang WB, Wightman R, Meyerowitz EM (2017). Cell cycle control by nuclear sequestration of CDC20 and CDH1 mRNA in plant stem cells. Mol Cell 68, 1108-1119. DOI URL
53	Yu Y, Zhang YC, Chen XM, Chen YQ (2019). Plant noncoding RNAs: hidden players in development and stress responses. Annu Rev Cell Dev Biol 35, 407-431. DOI
54	Zhang C, Fan LS, Le BH, Ye PY, Mo BX, Chen XM (2020). Regulation of ARGONAUTE10 expression enables temporal and spatial precision in axillary meristem initiation in Arabidopsis. Dev Cell 55, 603-616. DOI URL
55	Zhang C, Wang J, Wenkel S, Chandler JW, Werr W, Jiao YL (2018). Spatiotemporal control of axillary meristem formation by interacting transcriptional regulators. Development 145, dev158352.

[1]	CHEN Xin-Rui, SONG Wei-Feng, Yi WANG, Hao WANG, Shi-Yao SUN, Cai-Jiang WANG, Shi-Peng CAI, Hong REN, Yu-Tao HE, Min PAN, Guang-Xiu CAO, Yi YAN, Zhi-Yong XIE, WANG Hang. Nitrogen and phosphorus resorption characteristics and adaptive strategies of typical emergent plants in the lakeshore zone of Dianchi Lake, China [J]. , 2026, 50(预发表): 0-.
[2]	XI Nian-Xun. The impact of multiple global change factors on traits of mycorrhizal plants [J]. , 2026, 50(预发表): 0-.
[3]	Chenyang Hao, Shaoyu Gao, Yuehua Cheng, Zhaxinima, Bo Xu, Yang Yang. Ecological significance of the compact and dark inflorescence of Saussurea velutina (Asteraceae) in Hengduan Mountains, Southwestern China [J]. Biodiv Sci, 2026, 34(5): 25489-.
[4]	Xiansheng Chen, Yuchen Wang, Hongliang Zhang, Bo Peng, Yingli Wang, Yuan Huang. Advances in Plant Wearable Sensors for the Physiological and Biochemical Information of Horticultural Crops [J]. Chinese Bulletin of Botany, 2026, 61(4): 1-0.
[5]	Xiao-Yong Chen. On the effective ex situ conservation of rare or endangered plants: Definition, standards and recommendations [J]. Biodiv Sci, 2026, 34(4): 25448-.
[6]	Mingtai An, Xu Wu, Jianghong Yu, Guoxiong Hu³, Kun Wang, Feng Liu, Tianrou Wu, Qin Xu, Hao Zhang, Jian Xu, Yanbing Yang, Wei Cao, Lingbin Yan, He Li, Zhi Li, Dehui Yu, Lang Huang, Zhiping Chen, Xiaofang Li, Huakai Zou, Min Long, Weihao Gu, Yuhang Ma, Jian Qiu. A dataset on inventory and geographical distributions of higher plants in Guizhou, China [J]. Biodiv Sci, 2026, 34(4): 25427-.
[7]	Shengyun Wang, Erbiao Guo, Mengjun Li, Zhonghua Wang. Leaf Color Mutants in Medicinal Plants: Induction and Mutation Mechanisms [J]. Chinese Bulletin of Botany, 2026, 61(3): 529-540.
[8]	Liangliang Zhang, Xianting Wang, Yong Chen, Yifan Zhu, Xinyuan Lu, Zaitseva Svetlana Mikhailovna, Haiyun Yang. Establishment of Tissue Culture and Rapid Propagation System of Wild Plant Parrotia subaequalis Under National First Class Protection [J]. Chinese Bulletin of Botany, 2026, 61(1): 114-122.
[9]	Xinyi Guo, Zhen Yang, Jia Wang, Yanbing Lin, Zhouping Shangguan, Miaochun Fan. The Exploration and Application Potential of Rhizosphere-promoting Bacterial Resources from Native Leguminous Plants in Arid Regions of China [J]. Chinese Bulletin of Botany, 2026, 61(1): 170-178.
[10]	GUO Zhi-Hong, YANG Ni, ZHANG Tao, LI Hai-Bo, TIAN Tai-An, HUANG Xiao-Bo, LI Cong, MA Si-Ju, SU Jian-Rong, LI Shuai-Feng. Change of functional diversity and community assembly of mycorrhizal plant along an altitudinal gradient in primary forests of Fanjing Mountain, Guizhou, China [J]. Chin J Plant Ecol, 2025, 49(9): 1410-1423.
[11]	Shengnan Ji, Jiarong Han, Yueheng Ren, Xiaodong Mu, Yanpeng Zhu. Prediction of suitable habitats and risk assessment for key invasive alien plant species on Hainan Island based on the MaxEnt model [J]. Biodiv Sci, 2025, 33(8): 25037-.
[12]	CUI Dong-Qing, TIAN Chen, SONG Hui-Min, LU Xiao-Ming, SA Qi-Ri, XU Guo-Qing, YANG Pei-Zhi, BAI Yong-Fei, TIAN Jian-Qing. Response mechanisms of rhizosphere bacterial community diversity and functional group composition of dominant plants in typical grasslands to long-term grazing [J]. Chin J Plant Ecol, 2025, 49(7): 1163-1176.
[13]	Shuai Li, Weihua Liu, Yudan Xu, Xiaobo Tian, Houjuan Song, Xiaoting Yue, Lingling Wu, Qing Zhang, Tieliang Shanguan. A dataset on inventory and geographical distributions of vascular plants in Shanxi, China [J]. Biodiv Sci, 2025, 33(7): 24317-.
[14]	Mengsha Huang, Lingdie Kong, Miao Yu, Chang Liu, Siqin Wang, Ruohan Wang. Development and Application of 3D Reconstruction Technology at Different Scales in Plant Research [J]. Chinese Bulletin of Botany, 2025, 60(6): 1005-1016.
[15]	Xu Yufeng, Zhou Mian. Post-transcriptional Regulation in Plant Immunity [J]. Chinese Bulletin of Botany, 2025, 60(5): 704-721.