Effect of overexpression of IPW on RNA expression in fetal RPE

To reveal the clinical significance, pathological involvement and molecular mechanism of IPW in retinal pigment epithelium anomalies that contribute to age-related macular degeneration. Microarrays were performed to detect IPW expression under pathological conditions. RNA was extracted with its concentration and purity measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and its integrity assessed using the RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). A total amount of 1 μg RNA was sent for sequencing library generation using the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology, Shanghai, China) following the manufacturer’s recommendations. The library fragments were then purified with the AMPure XP system (Beckman Coulter), and sequenced on an Illumina NovaSeq platform to generate 150 bp paired-end reads, according to the manufacturer’s instructions.

本研究旨在揭示IPW在与年龄相关性黄斑变性（age-related macular degeneration）相关的视网膜色素上皮（retinal pigment epithelium）异常中的临床意义、病理累及特征及分子机制。本研究通过基因芯片技术检测病理状态下IPW的表达水平。提取RNA后，采用NanoDrop 2000分光光度计（赛默飞世尔科技，Thermo Fisher Scientific）检测其浓度与纯度，并通过安捷伦生物分析仪2100系统（Agilent Technologies，美国加利福尼亚州圣克拉拉市安捷伦科技公司）搭载的RNA Nano 6000检测试剂盒评估其完整性。取总量为1 μg的RNA，按照厂商推荐的操作流程，使用适配Illumina平台的Hieff NGS Ultima双模式mRNA文库制备试剂盒（上海翌圣生物科技有限公司，Yeasen Biotechnology，中国上海）构建测序文库。随后采用AMPure XP磁珠纯化系统（贝克曼库尔特，Beckman Coulter）纯化文库片段，并依照厂商说明在Illumina NovaSeq测序平台上进行测序，生成150 bp双端读段（paired-end reads）。