Evaluating Gene Expression in C57BL/6J and DBA/2J Mouse Striatum Using RNA-Seq and Microarray

C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, 'digital mRNA counting' is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation. There are 5 male and 5 female C57BL/6J animals and 5 male and 5 female DBA/2J. These are adult, naive animals. One array is run for each animal for a total of 20 Mouse MOE430 2.0 arrays. The purpose is to determine baseline (naive) gene expression differences in striatum for these two inbred strains. There are 12 male C57BL/6J animals and 12 male DBA/2J. These are adult, naive animals. One array is run for each animal for a total of 24 Mouse Ref8 v2 arrays. The purpose is to determine baseline (naive) gene expression differences in striatum for these two inbred strains. ***This submission represents the microarray component of the studyC57BL/6J（B6）与DBA/2J（D2）是神经科学研究中最常用的两种近交系小鼠品系。然而，目前唯一可用的小鼠基因组完全基于B6品系的序列。此后开发的寡核苷酸微阵列探针仅依托该B6参考序列设计，由于两品系间存在包括单核苷酸多态性（single nucleotide polymorphisms, SNPs）在内的等位基因序列差异，利用此类探针开展跨品系基因表达谱比较的可靠性存疑。 下一代测序（next-generation sequencing, NGS）技术与RNA测序（RNA-Seq）的出现，为检测差异基因表达提供了替代寡核苷酸芯片的理想方案，可规避基于杂交技术的固有缺陷。本研究共纳入21个样本（10个B6样本、11个D2样本），每个样本平均生成2200万条短测序reads，将这些reads比对至小鼠参考基因组后，可在两个品系的纹状体中对16183个Ensembl基因进行表达量查询。为确定差异表达基因，本研究基于比对至外显子的reads应用「数字mRNA计数」法。 本研究将RNA-Seq（Illumina GA IIx平台）与两种微阵列平台（Illumina MouseRef-8 v2.0和Affymetrix MOE 430 2.0）进行对比，以检测B6与D2近交系小鼠纹状体的基因表达差异。结果显示，在严格的数据处理要求下，RNA-Seq检测到的差异表达结果与Affymetrix、Illumina两个平台均一致的案例，多于仅与单个平台一致的案例，且折叠变化方向不一致的案例极为罕见。此外，相较于基于杂交的技术，RNA-Seq可获取更多信息：其检出的基因数量多于任一微阵列平台。绝大多数在RNA-Seq中检测到的差异表达基因仅能被RNA-Seq单独检出，这对于效应量较小的研究至关重要——此类研究中基于杂交技术的灵敏度可能会偏倚结果解读。 本研究第一组样本包含5只雄性与5只雌性C57BL/6J动物、5只雄性与5只雌性DBA/2J动物，均为成年未处理动物（naive animals）。每只动物对应一张Mouse MOE 430 2.0芯片，共计20张此类芯片，实验目的为确定这两个近交系小鼠纹状体的基线基因表达差异。第二组样本包含12只雄性C57BL/6J与12只雄性DBA/2J成年未处理动物，每只动物对应一张Illumina MouseRef-8 v2.0芯片，共计24张此类芯片，实验目的同样为确定这两个近交系小鼠纹状体的基线基因表达差异。***本提交内容对应本研究的微阵列部分。