Scaling the ChIP-chip assay to improve analysis of clinical biospecimens

扩展 ChIP 芯片检测以改进临床生物样本的分析

• 批准号: 7442311
• 负责人: PEGGY J Farnham
• 金额: $ 19万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7442311
• 关键词: Admixture; Antibodies; Biological Assay; Biological Markers; Cell Count; Cells; Chromatin; Clinical; Collection; Complex; Cultured Cells; DNA; Diagnosis; Epigenetic Process; Freezing; Gene Dosage; Gene Expression; Gene Silencing; Genetic Transcription; Genome; Genomics; Histone H3; Human; Lasers; Magic; Mediating; Methods; Microscopy; Miniaturization; Modification; Molecular; Normal Cell; Numbers; Pattern; Performance; Population; Preparation; Process; Protocols documentation; Research Personnel; Sampling; Series; Techniques; Tissues; base; cancer genome; improved; insight; neoplastic cell; novel therapeutics; succinylated wheat germ agglutinin; therapeutic target; tumor

DESCRIPTION (provided by applicant): Identifying gene expression changes that occur when a normal cell transforms into a neoplastic cell can reveal molecular markers that can be used for diagnosis and suggest potential new therapeutic targets. However, by characterizing the mechanisms by which the tumor-specific gene expression patterns have been created, one can also gain insight into the molecular processes that drive tumor formation. We have developed a protocol (termed a MAGIC analysis), based on the technique of ChIP-chip, that can be used both to study altered gene expression and to determine if the altered gene expression is due to changes in transcription complex formation, epigenetic-mediated gene silencing, or alterations in gene copy number. This method uses an antibody to RNAPII, an antibody to modified Histone H3, and an antibody to methylated DNA to identify active vs. silent regions of the genome. It also employs a genomic comparison of the normal and tumor samples to identify amplified and deleted regions of the genome. Most investigators who perform ChIP-chip assays use cultured cells. Clearly, this technique would be much more useful for characterizing the cancer genome if pure populations of tumor cells, obtained by microdisssection, laser capture, or other separation methods were analyzed. Adapting the ChIP-chip technique for the study of such samples requires modifications to sample preparation, scaling the ChIP assay to accommodate a small number of cells, and improving the amplification of the ChIP sample such that accurate representation of the experimental and control samples can be maintained when using small amounts of input DNA. We propose to modify the ChIP protocol for use with very small numbers of cells (Aim I), to determine the optimal amplification method for ChIP samples obtained from a small number of cells (Aim II), and to demonstrate that the modified protocols are suitable for analysis of cells obtained from tumor samples by laser capture microscopy (Aim III). We propose to modify the technique of ChIP-chip such that it can be used after collection of a small number of tumor cells by laser capture microscopy. Such modifications would be a great advance in the molecular characterization of the cancer genome.

描述（由申请人提供）：鉴定正常细胞转化为肿瘤细胞时发生的基因表达变化可以揭示可用于诊断的分子标志物，并提示潜在的新治疗靶点。然而，通过表征肿瘤特异性基因表达模式产生的机制，人们也可以深入了解驱动肿瘤形成的分子过程。我们开发了一种基于ChIP芯片技术的方案（称为MAGIC分析），可用于研究基因表达的改变，并确定基因表达的改变是否是由于转录复合物形成的变化、表观遗传介导的基因沉默或基因拷贝数的改变。该方法使用RNAPII的抗体、修饰的组蛋白H3的抗体和甲基化DNA的抗体来鉴定基因组的活性与沉默区域。它还采用正常和肿瘤样本的基因组比较来识别基因组的扩增和缺失区域。大多数进行ChIP芯片检测的研究人员使用培养的细胞。显然，如果对通过显微切割、激光捕获或其他分离方法获得的纯肿瘤细胞群进行分析，这种技术将对表征癌症基因组更有用。将ChIP芯片技术用于此类样品的研究需要修改样品制备，缩放ChIP测定以适应少量细胞，并改善ChIP样品的扩增，使得当使用少量输入DNA时可以保持实验和对照样品的准确表示。我们建议修改的ChIP协议使用非常少的细胞（目的I），以确定最佳的扩增方法从少量的细胞（目的II）获得的ChIP样品，并证明修改后的协议是适合于分析从肿瘤样品中获得的细胞激光捕获显微镜（目的III）。我们建议修改ChIP芯片的技术，使其可以在通过激光捕获显微镜收集少量肿瘤细胞后使用。这种修饰将是癌症基因组分子特征的一大进步。