Martoglio A M, Tom B D, Starkey M, Corps A N, Charnock-Jones D S, Smith S K
Department of Obstetrics and Gynaecology, University of Cambridge, England.
Mol Med. 2000 Sep;6(9):750-65.
Complementary DNA array analysis of gene expression has a potential application for clinical diagnosis of disease processes. However, accessibility, affordability, reproducibility of results, and management of the data generated remain issues of concern. Use of cDNA arrays tailored for studies of specific pathways, tissues, or disease states may render a cost- and time-effective method to define potential hallmark genotype alterations.
We produced a 332-membered human cDNA array on nylon membranes tailored for studies of angiogenesis and tumorigenesis in reproductive disease. We tested the system for reproducibility using a novel statistical approach for analysis of array data and employed the arrays to investigate gene expression alterations in ovarian cancer.
Intra-assay analysis and removal of agreement outliers was shown to be a critical step prior to interpretation of cDNA array data. The system revealed highly reproducible results, with intermembrane coefficient of reproducibility of +/- 0.98. Comparison of placental and ovarian sample data confirmed expected differences in angiogenic profiles and tissue-specific markers, such as human placental lactogen (hPL). Analysis of expression profiles of five normal ovary and four poorly differentiated serous papillary ovarian adenocarcinoma samples revealed an overall increase in angiogenesis-related markers, including vascular endothelial growth factor (VEGF) and angiopoietin-1 in the diseased tissue. These were accompanied by increases in immune response mediators (e.g. HLA-DR, Ron), apoptotic and neoplastic markers (e.g. BAD protein, b-myb), and novel potential markers of ovarian cancer, such as cofilin, moesin, and neuron-restrictive silencer factor (REST) protein.
In-house production of tailored cDNA arrays, coupled to comprehensive analysis of resulting hybridization profiles, provides an accessible, reliable, and highly effective method of applying array technology to study disease processes. In the ovary, abundance of specific tumor markers, increased macrophage recruitment mediators, a late-stage angiogenesis profile, and the presence of chemoresistance-related markers distinguished normal and advanced ovarian cancer tissue samples. Detection of such parallel changes in pathway- and tissue-specific markers may prove a hallmark ready for application in reproductive disease diagnostic and therapeutic developments.
基因表达的互补DNA阵列分析在疾病过程的临床诊断中具有潜在应用价值。然而，数据的可获取性、成本、结果的可重复性以及所产生数据的管理仍然是令人关注的问题。使用针对特定途径、组织或疾病状态研究定制的cDNA阵列，可能会提供一种经济高效的方法来确定潜在的标志性基因型改变。
我们在尼龙膜上制作了一个包含332个成员的人类cDNA阵列，该阵列专为生殖疾病中的血管生成和肿瘤发生研究而定制。我们使用一种新颖的统计方法分析阵列数据来测试该系统的可重复性，并使用这些阵列研究卵巢癌中的基因表达改变。
在解释cDNA阵列数据之前，实验内分析和去除一致性异常值被证明是关键步骤。该系统显示出高度可重复的结果，膜间可重复性系数为±0.98。胎盘和卵巢样本数据的比较证实了血管生成谱和组织特异性标志物（如人胎盘催乳素（hPL））的预期差异。对五个正常卵巢和四个低分化浆液性乳头状卵巢腺癌样本的表达谱分析显示，患病组织中血管生成相关标志物总体增加，包括血管内皮生长因子（VEGF）和血管生成素-1。这些变化伴随着免疫反应介质（如HLA-DR、Ron）、凋亡和肿瘤标志物（如BAD蛋白、b-myb）以及卵巢癌新的潜在标志物（如丝切蛋白、埃兹蛋白和神经元限制性沉默因子（REST）蛋白）的增加。
自行制作定制的cDNA阵列，并结合对所得杂交谱的综合分析，为应用阵列技术研究疾病过程提供了一种可获取、可靠且高效的方法。在卵巢中，特定肿瘤标志物的丰富、巨噬细胞募集介质的增加、晚期血管生成谱以及化疗耐药相关标志物的存在区分了正常和晚期卵巢癌组织样本。检测途径和组织特异性标志物中的此类平行变化可能证明是一种可用于生殖疾病诊断和治疗发展的标志性特征。
