Detection of mRNA and microRNA Expression in Basal Chordates, Amphioxus and Ascidians
This chapter describes whole-mount in situ hybridization protocols for the localization of mRNA transcripts on two closest invertebrate relatives of the vertebrates, cephalochordates (amphioxus) and urochordates (ascidians). The technique is based on the use of digoxigenin or fluorescein RNA probes for nonradioactive detection of transcripts. Then, the signal is visualized by differently colored water-insoluble precipitates using the indirect alkaline phosphatase-based chromogenic detection method. Such protocol may be also used with one or two colors in order to detect one or two different transcripts in the same preparation by double in situ hybridization. Our protocol is also optimized for the detection of microRNA transcripts based upon the use of LNA probes. In this chapter, we provide also a detailed method for detection of transcripts in resin sections of hybridized embryos. In conclusion, the method described in this chapter permits the detailed visualization of gene expression at single-cell resolution and makes it a useful tool for analyzing expression patterns of neuron specific genes in both amphioxus and ascidians.
Key wordsmRNA and microRNA expression Whole-mount ISH Amphioxus Ascidians Resin sections Double-ISH LNA probes Development
We would like to thank Skip Pierce and John M. Lawrence (Department of Biology, USF, Tampa, FL) for the use of laboratory space and equipment; Linda Holland and Nicholas Holland (Scripps Institution of Oceanography, La Jolla, CA) for the important support in collecting Branchiostoma floridae adults and embryos, and Héctor Escrivà and collaborators (Laboratoire de Biologie Intégrative des Organismes Marins (BIOM) UMR7232 CNRS-INSB-UPMC, Observatoire Océanologique de Banyuls-sur-Mer, 66650 Banyuls-sur-Mer, France) for the important support in collecting Branchiostoma lanceolatum adults and embryos. This work was supported by MIUR (PRIN Program no. 20088JEHW3-001) (to SC and MP) and by ASSEMBLE grant (no. 227799) (to SC, MP, and GG).
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