Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Stk11-/- Endothelial cell Transcriptomes [LKB1]

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: Endothelial cell mRNA profiles of 2-months-old wild-type (WT) and endothelial cell specific Serine/Threonine Kinase 11 (Stk11-/-) knock out mice 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