Abstract
A key element to understand developmental and reproductive processes like germline development, double fertilization, and embryogenesis is the study of cell-specific gene expression patterns which is best analyzed by RNA in situ hybridization. Different visualization techniques have been established to mark either the region of mRNA production (using the classical chromogenic detection system) or the specific localization of mRNAs (using fluorescent labeled probes). In this chapter, we describe and compare whole mount RNA in situ hybridization techniques on ovules and young developing seeds from Arabidopsis thaliana using three different detection systems. The alkaline phosphatase (AP) coupled antibody detecting the antigen labeled probe facilitates the production of a precipitating dye indicating mRNA presence: (1) using BCIP/NBT as substrates, it is converted to a blue staining that can be visualized using differential interference contrast (DIC) microscopy. Alternatively, (2) using Fast-Red as a substrate it is converted to a purple fluorescent staining that can be visualized either by light microscopy or, for a higher cellular resolution, by confocal microscope. To analyze mRNA distribution with subcellular resolution we (3) describe a third, highly sensitive fluorescent detection system, which is based on the enzymatic activity of a peroxidase. In combination with a tyramide signal amplification (TSA) system, it leads to multi-fluorescent labeled antibodies marking the mRNA bound probe locally.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Medioni C, Mowry K, Besse F (2012) Principles and roles of mRNA localization in animal development. Development 139:3263–3276. doi:10.1242/dev.078626
Buchan JR (2014) mRNP granules. RNA Biol 11:1019–1030. doi:10.4161/15476286.2014.972208
Notaguchi M, Higashiyama T, Suzuki T (2015) Identification of mRNAs that move over long distances using an RNA-seq analysis of Arabidopsis/Nicotiana benthamiana heterografts. Plant Cell Physiol 56:311–321. doi:10.1093/pcp/pcu210
Thieme CJ, Rojas-Triana M, Stecyk E et al (2015) Endogenous Arabidopsis messenger RNAs transported to distant tissues. Nat Plants 1:15025. doi:10.1038/nplants.2015.25
Zhang W, Thieme C, Kollwig G et al (2016) tRNA-related sequences trigger systemic mRNA transport in plants. Plant Cell 2016:tpc.01056.2015. doi:10.1105/tpc.15.01056
Hejátko J, Blilou I, Brewer PB et al (2006) In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples. Nat Protoc 1:1939–1946. doi:10.1038/nprot.2006.333
Jackson D (1991) In situ hybridization in plants. In: Bowles DJ, Gurr SJ, McPherson M (eds) Molecular plant pathology: a pract approach, series 1. Oxford University Press, Oxford, pp 163–174
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:10.1016/j.ymeth.2015.10.019
Acknowledgment
This study was funded by the Collaborate Research Center SFB960 of the German Research Foundation (DFG—Germany) to T.D.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Bleckmann, A., Dresselhaus, T. (2017). Whole Mount RNA-FISH on Ovules and Developing Seeds. In: Schmidt, A. (eds) Plant Germline Development. Methods in Molecular Biology, vol 1669. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7286-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7286-9_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7285-2
Online ISBN: 978-1-4939-7286-9
eBook Packages: Springer Protocols