High-throughput transcriptomics platform for screening hepatotoxicants

We introduce a new high-throughput transcriptomics (HTTr) platform comprised of a collagen sandwich primary rat hepatocyte culture and the TempO-Seq assay for screening and prioritizing potential hepatotoxicants. We selected 14 chemicals based on their risk of drug-induced liver injury (DILI) and tested them in hepatocytes at two treatment concentrations. HTTr data was generated using the TempO-Seq whole transcriptome and S1500+ assays. The HTTr platform exhibited high reproducibility between technical replicates (r>0.9) but biological replication was greater for TempO-Seq S1500+ (r>0.85) than for the whole transcriptome (r>0.7). Reproducibility between biological replicates was dependent on the strength of transcriptional effects induced by a chemical treatment. Despite targeting a smaller number of genes, the S1500+ assay clustered chemical treatments and produced gene set enrichment analysis (GSEA) scores comparable to those of the whole transcriptome. Connectivity mapping showed a high-level of reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project with high concordance between the S1500+ gene set and whole transcriptome. Taken together, our results provide guidance on selecting the number of technical and biological replicates and support the use of TempO-Seq S1500+ assay for a high-throughput platform for screening hepatotoxicants.我们介绍了一种新型高通量转录组学（high-throughput transcriptomics, HTTr）平台，该平台由胶原三明治法原代大鼠肝细胞培养体系与TempO-Seq检测技术构成，用于肝毒性潜在物质的筛选与优先级排序。本研究基于药物性肝损伤（drug-induced liver injury, DILI）风险筛选了14种化学物质，并在肝细胞中设置两种处理浓度开展测试。HTTr数据通过TempO-Seq全转录组检测与S1500+检测试剂盒生成。该HTTr平台的技术重复间呈现出极高的重复性（相关系数r>0.9）；但TempO-Seq S1500+检测的生物学重复相关性（r>0.85）优于全转录组检测（r>0.7）。生物学重复间的重复性水平取决于化学处理诱导的转录调控效应强度。尽管靶向基因数量更少，S1500+检测仍可实现化学处理样本的聚类分析，且生成的基因集富集分析（gene set enrichment analysis, GSEA）得分与全转录组检测结果相当。连接性图谱分析显示，TempO-Seq数据与Open TG-GATES项目中的Affymetrix基因芯片数据具有高度一致性与重复性，且S1500+基因集与全转录组基因集的匹配度较高。综上，本研究结果为技术重复与生物学重复的样本量选择提供了参考依据，并支持将TempO-Seq S1500+检测技术应用于肝毒性物质筛选的高通量研究平台。