Transcription profiling by high throughput sequencing in diaphysis and metaphysis from femur of 5-week-old Dlx3Oc-cKO and wild type mice

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived femoral diaphysis and metaphysis transcriptome profiling (RNA-seq) to determine pathways and networks dependent on Dlx3 during bone development and homeostasis. Methods: mRNA profiles of diaphysis and metaphysis isolated from the femur of 5-week-old wild-type (WT) and Dlx3Oc-cKO (OC-cre;Dlx3f/-) conditional knockout mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level by ANOVA (ANOVA) and TopHat. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq data were generated with Illumina's HiSeq 2000 system. Raw sequencing data were processed with CASAVA 1.8.2 to generate fastq files. Reads of 50 bases were mapped to the mouse transcriptome and genome mm9 using TopHat 1.3.2. Gene expression values (RPKM) were calculated with Partek Genomics Suite 6.6, which was also used for the ANOVA analysis to determine significantly differentially expressed genes. Conclusions: Our study represents the first detailed analysis of Dlx3Oc-cKO diaphysis and metaphysis from femurs, with biologic triplicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Diaphysis and metaphysis mRNA profiles of metaphysis and diaphysis from femurs of 5-wk-old (WT) and Dlx3Oc-cKO male mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.研究目的：下一代测序（Next-generation sequencing, NGS）革新了基于系统生物学的细胞通路分析。本研究旨在对比基于NGS得到的股骨骨干与干骺端转录组分析（RNA-sequencing, RNA-seq）结果，以明确骨骼发育与稳态过程中依赖Dlx3的通路及调控网络。 研究方法：从5周龄野生型（wild-type, WT）及Dlx3骨组织特异性条件性敲除小鼠（Dlx3Oc-cKO，即OC-cre;Dlx3f/-）的股骨中分离得到骨干与干骺端组织，通过Illumina HiSeq 2000平台进行深度测序，每组设置3次生物学重复。对通过质量过滤的序列读段，采用方差分析（Analysis of Variance, ANOVA）和TopHat工具在转录本异构体水平开展分析。采用SYBR Green法完成实时荧光定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）验证。 研究结果：本研究通过Illumina HiSeq 2000系统获取RNA-seq数据。原始测序数据经CASAVA 1.8.2处理后生成fastq格式文件。将50bp的序列读段比对至小鼠转录组及mm9版本参考基因组，比对工具采用TopHat 1.3.2。利用Partek Genomics Suite 6.6计算基因表达量（Reads Per Kilobase per Million mapped reads, RPKM），并通过该工具完成ANOVA分析以筛选显著差异表达基因。 研究结论：本研究首次针对Dlx3Oc-cKO小鼠股骨骨干与干骺端组织开展了详尽的转录组分析，所有样本均设置3次生物学重复，测序技术采用RNA-seq。本研究报道的优化数据分析流程可为表达谱的比较研究提供参考框架。我们认为，基于RNA-seq的转录组表征可加速遗传网络分析，并助力解析复杂的生物学功能。本次研究同样通过Illumina HiSeq 2000平台进行深度测序，设置3次生物学重复，获取了5周龄雄性野生型及Dlx3Oc-cKO小鼠股骨干骺端与骨干的mRNA表达谱。