Next Generation Sequencing Facilitates Quantitative Analysis of Gene expression profile in CD8+ T cells with TOX-overexpression

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived TOX associated 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 human CD8+ T cell overexpressed by TOX lentivirus or NC lentiviruswere generated by deep sequencing, in triplicate, using Illumina. qRT–PCR validation was performed using TaqMan and SYBR Green assays Conclusions: Our study represents the first detailed analysis of TOX associated transcriptomes generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Gene expression profile in CD8+ T cells with TOX-overexpression