Laser-Assisted Microdissection for Cell- and Tissue Type-Specific Transcriptome and RNA-Seq Analysis.

Monitoring gene activity at high spatial and temporal resolution is key to understanding the regulation of developmental programs. It allows researchers to uncover developmental trajectories in complex organs or tissues and to determine the expression of genes in rare cell or tissue types, such as the cells of the plant female reproductive lineages (germline), which are embedded within the sporophytic tissues of the flower. Investigations into plant germlines are of scientific interest in order to gain an understanding of transcriptional processes governing reproduction. Moreover, in general terms, plant germlines are well suited as models for investigating gene regulatory programs underlying organogenesis, as they are typically composed of only a small number of highly specialized cell types. In recent years, a number of transcriptome analyses have uncovered regulatory programs underlying major steps of germline development during both sexual and asexual reproduction through seeds (apomixis). Apomixis is of great interest, as it leads to a formation of clonal embryos by omission or alteration of meiosis by the cell specified as first cell of the female germline combined with parthenogenesis (absence of fertilization of the egg cell). To overcome the long-faced challenge of the inaccessibility of the cells for cell type-specific investigations, the establishment of laser-assisted microdissection (LAM) has been an important methodological advance. The method has been improved for isolation of high-quality RNA for RNA-Seq analyses for investigations on Arabidopsis thaliana and sexual and apomictic accessions from the closely related genus Boechera. In this chapter, the method from tissue fixation to RNA isolation is detailed.