Chowers Itay, Gunatilaka Tushara L, Farkas Ronald H, Qian Jiang, Hackam Abigail S, Duh Elia, Kageyama Masaaki, Wang Chenwei, Vora Amit, Campochiaro Peter A, Zack Donald J
Guerrieri Center at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Invest Ophthalmol Vis Sci. 2003 Sep;44(9):3732-41. doi: 10.1167/iovs.02-1080.
To construct a custom cDNA microarray for comprehensive human retinal gene expression profiling and apply it to the identification of genes that are preferentially expressed in the retina.
A cDNA microarray was constructed based on the predicted human retina gene expression profile according to expressed sequence tag (EST) databases. Gene expression profiles were obtained from five human retinas, two livers, and the cerebral cortical regions of two brains. Each sample was studied in duplicate, using a reference sample experimental design. Retina-enriched genes were identified by using the significance analysis for microarray (SAM) algorithm. Quantitative real time PCR was used to confirm microarray results. Bioinformatic analysis was performed to compare the array results with expression data available from public databases.
The cDNA microarray contains 10,034 sequences: 67% represent known genes and 33% represent ESTs. Differential hybridization with the array identified, in addition to known retinal genes, 186 retina-enriched genes that do not have known retinal function. Of these, 96 represent novel genes. Quantitative real-time PCR of 11 of the identified genes and ESTs confirmed their retina-enriched expression pattern. Bioinformatic analysis of EST databases suggests that of the 186 genes, approximately 40% are predominantly expressed in the retina, whereas the remainder show significant expression in other tissues. Comparison of this study's microarray-based retina-enriched gene set with three published similar sets identified using complementary high-throughput approaches demonstrated only limited overlap of the identified genes.
Because previous studies have demonstrated that many retina-enriched genes are crucial for maintaining normal retinal function, the genes identified here are likely to include ones that have important roles in the retina and ones that when mutated can cause or modulate retinal disease. In addition, the retina custom array should provide a useful resource for comparing expression profiles between normal and diseased human retinas.
构建定制的cDNA微阵列用于全面的人类视网膜基因表达谱分析，并将其应用于鉴定在视网膜中优先表达的基因。
根据表达序列标签（EST）数据库中预测的人类视网膜基因表达谱构建cDNA微阵列。从五个人类视网膜、两个肝脏和两个大脑的大脑皮质区域获得基因表达谱。每个样本使用参考样本实验设计进行重复研究。通过微阵列显著性分析（SAM）算法鉴定视网膜富集基因。使用定量实时PCR确认微阵列结果。进行生物信息学分析以将阵列结果与公共数据库中可用的表达数据进行比较。
该cDNA微阵列包含10,034个序列：67%代表已知基因，33%代表EST。与该阵列的差异杂交除了鉴定出已知的视网膜基因外，还发现了186个没有已知视网膜功能的视网膜富集基因。其中，96个代表新基因。对11个鉴定出的基因和EST进行定量实时PCR证实了它们在视网膜中的富集表达模式。对EST数据库的生物信息学分析表明，在这186个基因中，约40%主要在视网膜中表达，而其余基因在其他组织中也有显著表达。将本研究基于微阵列的视网膜富集基因集与使用互补高通量方法鉴定的三个已发表的类似基因集进行比较，发现鉴定出的基因只有有限的重叠。
由于先前的研究表明许多视网膜富集基因对于维持正常视网膜功能至关重要，因此这里鉴定出的基因可能包括在视网膜中起重要作用的基因以及那些突变时可导致或调节视网膜疾病的基因。此外，视网膜定制阵列应为比较正常和患病人类视网膜之间的表达谱提供有用的资源。
