Next Generation Sequencing Facilitates Quantitative Analysis of Wild and Myocardial ischemia-reperfusion injury

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived myocardial 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: Retinal mRNA profiles of 21-day-old Wild and myocardial ischemia-reperfusion injury 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

研究目的：下一代测序（Next-generation sequencing, NGS）已革新了细胞通路的系统生物学分析体系。本研究旨在对比基于NGS的心肌转录组分析（RNA-seq）与微阵列（microarray）、定量逆转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction, qRT–PCR）三种技术的应用效果，并优化适用于高通量数据分析的最优实验方案。实验方法：选取21日龄野生型小鼠与心肌缺血再灌注损伤小鼠，采用Illumina GAIIx平台完成三次生物学重复的深度测序，获取其视网膜mRNA表达谱。对通过质量过滤的序列读段，分别采用两种分析流程在转录本异构体水平开展数据分析：一是Burrows–Wheeler Aligner（BWA）结合方差分析（ANOVA），二是TopHat结合Cufflinks。此外采用TaqMan与SYBR Green检测方法完成qRT–PCR验证实验。