Next Generation Sequencing Facilitates Quantitative Analysis of Normal Human Kidney Transcriptomes. Homo sapiens

Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived normal human kidney 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 Overall design: The kidney tissue was immediately placed and stored in RNAlater® (Ambion), according to the manufacturer’s instruction. The tissue was manually microdissected under microscope in RNAlater® pool for glomerular and tubular compartment. Dissected tissue was homogenized and RNA was prepared using RNAeasy mini columns (Qiagen, Valencia, CA, US), according to the manufacturer’s instructions. RNA quality and quantity were determined using the Laboratory-on-Chip Total RNA PicoKit Agilent BioAnalyzer. Only samples without evidence of degradation were further used (RNA Integrity Number >6).

下一代测序（Next-generation Sequencing，NGS）已彻底革新了细胞通路的系统级分析。本研究旨在对比基于NGS的正常人肾转录组谱分析（RNA测序，RNA-seq）与微阵列、定量反转录聚合酶链反应（qRT–PCR）技术，并评估适用于最优高通量数据分析的实验方案。总体实验设计：按照制造商的操作说明，将肾脏组织立即置入RNAlater®（Ambion公司）中保存。在RNAlater®溶液中通过显微镜手动显微切割，分离获取肾小球与肾小管区域的组织。依照制造商的操作指南，对切割获取的组织进行匀浆处理，并使用RNAeasy迷你柱（Qiagen公司，美国加利福尼亚州巴伦西亚）提取总RNA。采用安捷伦生物分析仪配套的芯片实验室（Laboratory-on-Chip）总RNA PicoKit检测RNA的质量与浓度。仅选取无降解迹象的样本（RNA完整性指数>6）用于后续实验。