Novel approach to select genes from RMA normalized microarray data using functional hearing tests in aging mice

Presbycusis - age-related hearing loss - is the number one communicative disorder of our aged population. Here we analyzed gene expression for a set of GABA receptors in the cochlea of aging CBA mice using the Affymetrix GeneChip MOE430A. Functional phenotypic hearing measures distortion-product otoacoustic emission (DPOAE) amplitudes (four age groups) were made. The gene expression changes from RMA normalized microarray data (40 replicates) were first subjected to one-way ANOVA, and then linear regression was performed. In addition, the log signal ratio was converted to fold change, and selected gene expression changes were confirmed by relative real-time PCR. Major findings: expression of GABA-A receptor subunit 6was upregulated with age and hearing loss, whereas subunit 1 was repressed. In addition, GABA-A receptor associated protein like-1 and GABA-A receptor associated protein like-2 were strongly downregulated with age and hearing impairment. Lastly, gene expression measures were correlated with pathway/network relationships relevant to the inner ear using Pathway Architect, to identify key pathways consistent with the gene expression changes observed. In the study of expression changes GABA receptors in the in cochlea of young adult and aging presbycusis mice total of forty chips were used. The normal aging mice were in four groups young adults controls with good hearing (8 mice, 8 MOE430A GeneChips), Middle aged group with good hearing ( 17 mice, 17 MOE430A GeneChips), Mild Presbycusis (old) with limited hearing loss (9 mice, 9 MOE430A GeneChips) and Severe Presbycusis (old) (6 mice, 6 MOE430A GeneChips). Each Mice cochlea to each GeneChips, Samples was not pooled. The hearing potential evidence of each mouse is accompanied with each mice DPOAE amplitude.老年性聋（Presbycusis）即年龄相关性听力损失（age-related hearing loss），是老年人群中最常见的交流障碍性疾病。本研究使用Affymetrix GeneChip MOE430A基因芯片，对衰老CBA小鼠耳蜗内的一组γ-氨基丁酸（GABA）受体的基因表达谱进行了分析。同时针对四个年龄组小鼠开展了功能性听力表型检测，测定其畸变产物耳声发射（distortion-product otoacoustic emission, DPOAE）振幅。首先对经RMA标准化的微阵列数据（含40个生物学重复）中的基因表达变化进行单因素方差分析（one-way ANOVA），随后开展线性回归分析。此外，将对数信号比转换为表达倍数变化，并通过相对实时PCR（relative real-time PCR）对筛选出的差异表达基因进行验证。主要研究结果如下：GABA-A受体亚基6的表达随年龄增长及听力损失程度加重而上调，而亚基1的表达则被显著抑制。此外，GABA-A受体相关蛋白样1（GABA-A receptor associated protein like-1）与GABA-A受体相关蛋白样2的表达随年龄增长及听力损伤程度加剧而显著下调。最后，本研究使用Pathway Architect软件，将基因表达检测结果与内耳相关的通路/网络关联进行相关性分析，以鉴定与本次观测到的基因表达变化相符的关键通路。在针对年轻成年及衰老老年性聋小鼠耳蜗GABA受体表达变化的研究中，共使用了40张基因芯片。实验小鼠分为四组：听力正常的年轻成年对照组（8只小鼠，对应8张MOE430A基因芯片）、听力正常的中年组（17只小鼠，对应17张MOE430A基因芯片）、伴有轻度听力损失的老年性聋（老年）组（9只小鼠，对应9张MOE430A基因芯片），以及伴有重度听力损失的老年性聋（老年）组（6只小鼠，对应6张MOE430A基因芯片）。每只小鼠的耳蜗单独对应一张基因芯片，未进行样本混合。每只小鼠的听力检测结果均与其DPOAE振幅一一对应。