RNA-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

• Processo: 14/50396-0
• Modalidade de apoio: Auxílio à Pesquisa - Publicações científicas - Artigo
• Data de Início da vigência: 01 de junho de 2014
• Data de Término da vigência: 30 de novembro de 2014
• Área do conhecimento: Ciências Biológicas - Morfologia - Embriologia
• Pesquisador responsável: Chao Yun Irene Yan
• Beneficiário: Chao Yun Irene Yan
• Instituição Sede: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brasil
• Assunto(s): Análise de sequência de RNA Medula espinhal Embrião Pintos de corte Eletroporação Transcriptoma Publicações de divulgação científica Artigo científico
• Palavra(s)-Chave do Pesquisador: Chick Embryo | Galaxy Workflow | In Ovo Electroporation | Rna Seq | Spinal Cord | Transcriptome Profiling

Resumo

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. 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 instructions for electroporation and dissection of transfected spinal cord halves from HH22-23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatics 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 visual/y verified after comparison with control samples. (AU)