Detection of mRNA and microRNA expression in basal chordates, amphioxus and ascidians

Abstract

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.