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

描述(申请人提供)：识别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记，并提出潜在的新治疗靶点。然而，通过表征肿瘤特异性基因表达模式的产生机制，人们也可以深入了解驱动肿瘤形成的分子过程。我们已经开发了一种基于芯片技术的方法(称为魔术分析)，它既可以用来研究基因表达的变化，也可以用来确定基因表达的变化是由于转录复合体的变化、表观遗传介导的基因沉默还是基因拷贝数的变化。该方法使用针对RNAPII的抗体、针对修饰的组蛋白H3的抗体以及针对甲基化DNA的抗体来识别基因组的活跃区域和沉默区域。它还使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。大多数进行芯片分析的研究人员使用的是培养细胞。显然，如果对通过显微解剖、激光捕获或其他分离方法获得的纯肿瘤细胞群体进行分析，这项技术将对表征癌症基因组更加有用。将芯片技术应用于此类样品的研究需要对样品制备进行修改，调整芯片分析以适应少量细胞，并改进芯片样品的放大，以便在使用少量输入DNA时能够保持实验和对照样品的准确表示。我们建议修改芯片协议以用于非常少量的细胞(AIM I)，以确定从少量细胞获得的芯片样本的最佳扩增方法(AIM II)，并证明修改后的协议适用于通过激光捕获显微镜从肿瘤样本中获得的细胞的分析(AIM III)。我们建议对芯片技术进行改进，以便在激光捕获显微镜下采集少量肿瘤细胞后使用。这样的修饰将是癌症基因组分子特征方面的一大进步。

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.