Paez Gerardo L, Sellers Kimberly F, Band Mark, Acland Gregory M, Zangerl Barbara, Aguirre Gustavo D
Department of Clinical Studies--Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Mol Vis. 2006 Sep 7;12:1048-56.
Construction of a canine retinal custom cDNA microarray for comprehensive retinal gene expression profiling and application for the identification of genes that are preferentially expressed in the retina and brain lobes using a brain pool reference tissue.
A cDNA microarray was constructed utilizing clones obtained from a normalized canine retinal expressed sequence tag library. Gene expression profiles were analyzed for normal retina, as well as the cortex of the frontal, occipital, and temporal brain regions. Each sample was studied against a reference sample of pooled brain RNA. Data from a quantified scanned image were normalized using the loess subgrid procedure. Retina-enriched genes were identified using the Significance Analysis of Microarrays (SAM) algorithm, and confirmed by northern blot analyses for selected genes. Differences between biological samples were displayed using principal component analysis (PCA).
Expression profiles for each tissue set were analyzed against the common reference of pooled brain. Changes in expression between the sample and the reference were higher in the retina (27.9%) than the individual brain tissues (2-6.6%). Furthermore, all individual retinal samples were clearly separated from any of the hybridizations using brain tissue in the PCA. The accuracy of observed changes in expression has been confirmed by northern blot analysis using five randomly chosen genes that represented a wide range of different expression levels between retina and brain.
We have established an accurate and robust microarray system suitable for the investigation of expression patterns in the retina and brain. Characterization of the gene expression profiles in normal retina will facilitate the understanding of the processes that underline differences between normal and diseased retinas.
构建犬视网膜定制cDNA微阵列，用于全面的视网膜基因表达谱分析，并利用脑池参考组织鉴定在视网膜和脑叶中优先表达的基因。
利用从标准化犬视网膜表达序列标签文库中获得的克隆构建cDNA微阵列。分析正常视网膜以及额叶、枕叶和颞叶脑区皮质的基因表达谱。每个样本与合并脑RNA的参考样本进行比较研究。使用局部加权回归（loess）子网格程序对定量扫描图像的数据进行归一化处理。使用微阵列显著性分析（SAM）算法鉴定视网膜富集基因，并通过对选定基因的Northern印迹分析进行确认。使用主成分分析（PCA）展示生物样本之间的差异。
针对合并脑的共同参考分析每个组织集的表达谱。样本与参考之间的表达变化在视网膜中（27.9%）高于个体脑组织（2 - 6.6%）。此外，在主成分分析中，所有单个视网膜样本都与使用脑组织的任何杂交明显分开。通过对五个随机选择的基因进行Northern印迹分析，证实了观察到的表达变化的准确性，这些基因代表了视网膜和脑之间广泛不同的表达水平。
我们建立了一个准确且稳健的微阵列系统，适用于研究视网膜和脑中的表达模式。正常视网膜基因表达谱的表征将有助于理解正常视网膜与患病视网膜之间差异的潜在过程。
