Tissue specific RNA isolation in Drosophila embryos: a strategy to analyze context dependent transcriptome landscapes using FACS

Methods Mol Biol. 2014;1196:183-95. doi: 10.1007/978-1-4939-1242-1_11.

Abstract

The Hox family of transcription factors defines cell identity along the A/P axis of animal body plan by modulating expression of distinct sets of target genes in a tissue specific manner. Identifying such tissue specific target genes is indispensable if one wants to understand how Hox proteins mediate their context dependent function. Genome wide analysis of transcriptional activity in different tissues and contexts regarding Hox genes activity could help in reaching this goal. Such experiments rely on the possibility to selectively purify the cells of interest from developing embryos and to perform a transcriptomic analysis on such purified cell populations. By combining expression of a fluorescent protein and fluorescent activating cell sorting (FACS) technique, it is possible to obtain highly purified specific cell populations. In this chapter we describe the experimental procedure we have established in Drosophila-starting from a genetically marked small cell population (cardiomyocytes, 104 cells)-to dissociate the embryos in order to turn it into a suspension of individual cells, sort cells according to the expression of the introduced genetic marker and purify the total RNA content of the sorted cells. This can be used to analyze the transcriptome landscape of rare cell populations in wild type and mutant contexts. This technique has shown to be useful in the case of cardiac cells but is virtually applicable to any cell type and mutant backgrounds, provided that specific genetic markers are available.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Drosophila / genetics*
  • Drosophila / metabolism
  • Embryo, Nonmammalian / metabolism*
  • Flow Cytometry
  • Gene Expression Profiling
  • Organ Specificity / genetics
  • RNA / genetics
  • RNA / isolation & purification*
  • Transcriptome

Substances

  • RNA