Next Generation Sequencing Facilitates Quantitative Analysis of endothelial cells transfected with Ptbp1(wt), Ptbp1(T138A) or Ptbp1(T138E) plasmid Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived endothelial cell 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: mRNA profiles of endothelial cells transfected with Ptbp1(wt), Ptbp1(T138A) or Ptbp1(T138E) plasmid were generated 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