Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and atrn-/- brain Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the brain of atrn wildtype and homozygous adult zebrafish transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Total RNA was isolated from single brain of the wildtype and atrn-/- zebrafish using TRIzol reagent (Invitrogen) respectively by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Bioinformatics analysis of the transcriptome sequencing found that the steroid synthesis pathway was enriched in results of the mutant brain. By analyzing the data of male and female zebrafish with atrn mutations, it was found that the expression levels of related genes in the steroid synthesis pathway were up-regulated, mainly including the gene encoding the key enzyme for cholesterol synthesis, cyp51 and the gene encoding the key enzyme for testosterone synthesis, hsd17b7. Adult zebrafish brains of atrn+/+, atrn+/- and atrn-/- (4 month-post-fertilization)