title: RNA-Seq analysis of differential gene expression in electroporated chick embryonic spinal cord.
abstract: In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.
authors.affiliation: Department of Cell and Developmental Biology, Universidade de São Paulo; fmvieceli@usp.br.
authors.forename: Felipe M
authors.initials: FM
authors.lastname: Vieceli
authors.affiliation: Department of Cell and Developmental Biology, Universidade de São Paulo.
authors.forename: C Y Irene
authors.initials: CY
authors.lastname: Yan
mesh_terms: D000818:Animals; D002642:Chick Embryo; D004210:Dissection; D018274:Electroporation / Q000379:methods; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013116:Spinal Cord / Q000196:embryology / Q000378:metabolism / Q000502:physiology* / Q000601:surgery; D014162:Transfection / Q000379:methods