Development of a microarray chip for gene expression in rabbit ocular research
Development of a microarray chip for gene expression in rabbit ocular research
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发表时间：
2007-02
期刊：
影响因子：
2.2
通讯作者：
M. Popp;Li Liu;A. Timmers;D. Esson;L. Shiroma;C. Meyers;S. Berceli;M. Tao;G. Wistow;G. Schultz;M. Sherwood
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作者：
M. Popp;Li Liu;A. Timmers;D. Esson;L. Shiroma;C. Meyers;S. Berceli;M. Tao;G. Wistow;G. Schultz;M. Sherwood
目的建立一种可用于兔眼基因表达研究的基因芯片。方法从兔眼眼前段组织（角膜、结膜、虹膜）和后段组织（透镜、视网膜、巩膜）中提取mRNA，通过NEIBank工程建立两个独立的cDNA文库。从5'和3'端对来自这些文库中的每一个的克隆进行测序。将这些序列和来自美国国家生物技术信息中心（NCBI）兔子分类数据库的序列组合并电子组装成一组独特的非重叠连续序列（重叠群）。对于每一个重叠群，使用BLASTX和BLACKS针对NCBI NR和NT数据库进行同源性搜索以提供基因注释。将独特的重叠群发送到Agilent Technologies，其中在两个不同的阵列上以8阵列X 1900元件格式原位设计和合成60个碱基的寡核苷酸探针。对6只兔的一只眼进行青光眼滤过手术。14天后，从手术治疗和未治疗的对照眼的结膜和Tenon囊收获组织。来自每个样品的总RNA用花青染料标记并与我们的定制微阵列杂交。结果在两个阵列上存在的3，154个探针中，2，522个具有高于背景的信号值。青光眼滤过手术显著改变了315个基因的表达。表达改变的基因包括与炎症反应、防御反应相关的蛋白质和参与细胞外基质合成的蛋白质。结论本实验的结果与其他伤口愈合研究的结果一致，表明该芯片可以提供广泛的基因表达模式的有效信息。这是第一个可用于兔子研究的微阵列，是一个有价值的工具，可用于研究眼睛中的分子事件。
Purpose To develop a microarray for the rabbit that can be used for ocular gene expression research. Methods Messenger RNA was isolated from anterior segment tissues (cornea, conjunctiva, and iris) and posterior segment tissues (lens, retina, and sclera) of rabbit eyes and used to create two independent cDNA libraries through the NEIBank project. Clones from each of these libraries were sequenced from both the 5' and 3' ends. These sequences and those from the National Center for Biotechnology Information (NCBI) taxonomy database for rabbit were combined and electronically assembled into a set of unique nonoverlapping continuous sequences (contigs). For each contig, a homology search was performed using BLASTX and BLASTN against both the NCBI NR and NT databases to provide gene annotation. Unique contigs were sent to Agilent Technologies, where 60 base oligonucleotide probes were designed and synthesized, in situ, on two different arrays in an 8 array x 1900 element format. Glaucoma filtration surgery was performed on one eye of six rabbits. After 14 days, tissue was harvested from the conjunctiva and Tenon's capsule of both the surgically treated and untreated control eyes. Total RNA from each sample was labeled with cyanine dyes and hybridized to our custom microarrays. Results Of the 3,154 total probes present on the two arrays, 2,522 had a signal value above the background. The expression of 315 genes was significantly altered by glaucoma filtration surgery. Genes whose expression was altered included proteins associated with inflammatory response, defense response, and proteins involved in synthesis of the extracellular matrix. Conclusions The results of this rabbit microarray study are consistent with those from other wound healing studies, indicating that this array can provide valid information on broad patterns of gene expression. This is the first microarray available for rabbit studies and is a valuable tool that can be used to study molecular events in the eye.