Next generation sequencing facilitates quantitative analysis of tissue-specific molecular signatures in lupus mice

Purpose: Next-generation sequencing (NGS) technology was used to map expression profiles of effector (spleen) and key target tissues (kidney and brain) in mouse model of Systemic Lupus Erythematosus (SLE). Methods: Total RNA was extracted from total brain, total kidney and total spleen using Trizol and mRNA libraries were generated using the Illumina TruSeq Sample Preparation kit v2. Paired-end 37-bp mRNA sequencing was performed on Illumina HiSeq2000 platform. Quality of sequencing was assessed using FastQC software. Raw reads in fastq format were collected and aligned to the mouse genome (mm10 version) using STAR 2.6 algorithm. Gene quantification was performed using HTSeq and differential expression analysis was performed using edgeR package. Results: We defined kidney-specific molecular signatures of the murine lupus transcriptome that mirrors nephritis-specific transcriptome alterations in human SLE. Conclusions: By the use of the mouse kidney-specific transcriptome and through training of a large whole-blood RNA-sequencing dataset of SLE patients, we developed and validated an algorithm that predicts patients with active LN from SLE patients without LN and suggest vigilance in the monitoring of these patients and potential enrollment in LN prevention studies. New Zealand Black (NZB) female mice were crossed with New Zealand White (NZW) male mice. Female hybrids NZB/W-F1 (n=3 at each time-point) were sacrificed with perfusion with PBS at the pre-puberty stage (1 month), pre-autoimmunity stage (3 months) and nephritic stage (6 months old where proteinuria is more than 200 mg/dl for 3 consecutive days) of the disease. Spleen, kidneys and brain were extracted. Age-matched female C57BL/6 mice (n=3) were used as controls.研究目的：采用下一代测序（Next-generation sequencing, NGS）技术，绘制系统性红斑狼疮（Systemic Lupus Erythematosus, SLE）小鼠模型中效应组织（脾脏）与关键靶组织（肾脏、大脑）的表达谱。 研究方法：使用Trizol试剂从全脑、全肾及全脾中提取总RNA，采用Illumina TruSeq样本制备试剂盒v2构建mRNA文库。在Illumina HiSeq2000平台上完成双端37bp mRNA测序。使用FastQC软件评估测序质量。收集fastq格式的原始测序读段，通过STAR 2.6算法比对至小鼠基因组（mm10版本）。采用HTSeq进行基因定量分析，借助edgeR软件包完成差异表达分析。 研究结果：我们鉴定出小鼠狼疮转录组的肾脏特异性分子特征，该特征可反映人类SLE中肾炎特异性的转录组改变。 研究结论：通过利用小鼠肾脏特异性转录组，并结合大型SLE患者全血RNA测序数据集的训练，我们开发并验证了一种算法，该算法可区分活动性狼疮肾炎（Lupus Nephritis, LN）患者与无肾炎的SLE患者，并建议加强对这类患者的监测，以及考虑将其纳入狼疮肾炎预防研究。将新西兰黑鼠（New Zealand Black, NZB）雌性个体与新西兰白鼠（New Zealand White, NZW）雄性个体进行杂交。每个时间点取3只雌性杂交后代NZB/W-F1，分别在疾病的青春期前（1月龄）、自身免疫前（3月龄）及肾炎期（6月龄，即连续3天尿蛋白＞200mg/dl）通过磷酸盐缓冲液（Phosphate Buffered Saline, PBS）灌注处死后，采集脾脏、肾脏及大脑组织。设置年龄匹配的雌性C57BL/6小鼠（n=3）作为对照。