Analysis of gene expression changes in breast cancer using MALDI-TOF MS

描述(由申请人提供)：在这里，我们建议开发一种新的方法，用于基因表达水平变化的基因组分析。一组基因表达模式的变化被认为可以描述这些基因的生理功能，并可以阐明乳腺癌等复杂疾病在不同阶段的分子特征。因此，使用一种准确、快速和经济的方法有效地测量基因表达水平，将有助于了解乳腺癌的发展和疾病的诊断。该方法使用了两种方法，即固相可捕获单碱基延伸(SPC-SBE)方法和真实竞争PCR(RcPCR)方法。SPC-SBE是PI利用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)和分子亲和系统开发的一种多重基因分型方法。SPC-SBE方法包括在MS测量之前对DNA延伸片段进行特定分离的程序，允许在遗传变异的多重基因分型中获得更高的吞吐量和准确性。由于SPC-SBE为杂合子SNPs提供了一种可靠的基因分型方法，具有较高的准确度和吞吐量，因此我们推测，当采用rcPCR方法时，该方法可以成为一种稳健的基因表达分析方法。本研究的具体目的包括：(1)利用SPC-SBE和rcPCR技术分析50个基因的表达水平。我们将首先使用SPC-SBE和rcPCR开发一个表达分析工具。我们将使用一个研究得很好的系统，视网膜发育过程中基因表达的变化，作为建立所提出的方法的模型。(2)MALDI-TOF MS样品制备工艺的优化我们将通过优化样品晶体制备和数据分析来提高定量基因分型的准确性(从而提高基因表达水平的测量)。(3)乳腺癌样本检测系统的验证。我们将展示我们用于监测乳腺癌基因表达变化的系统。这一结果将与之前的研究进行比较。所提出的方法将允许高通量、准确和经济有效地监测乳腺癌基因表达水平的变化。因此，该方法将提供乳腺癌亚型的简单分类和进展阶段的准确确定，这对于个体患者评估和个性化治疗至关重要。

DESCRIPTION (provided by applicant): Here we propose to develop a new approach for genomic analysis of gene expression level changes. Changes in the expression pattern of a group of genes are believed to profile the physiological functions of those genes, and can illustrate the molecular characteristic of a complex disease such as breast cancer at various stages. Therefore, efficient measurements of gene expression level using an accurate, rapid and cost-effective way as proposed here will allow for the understanding of the breast cancer development and for the diagnosis of the disease. The proposed approach utilizes two methods, the solid phase capturable single base extension (SPC-SBE) method and the real competitive PCR (rcPCR) approach. SPC-SBE is a multiplex genotyping method developed by the PI that employs matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and a molecular affinity system. The SPC-SBE method includes a procedure for the specific isolation of DNA extension fragments prior to the MS measurement, allowing higher throughput and accuracy in multiplex genotyping of genetic variations. Since SPC-SBE offers a robust genotyping of heterozygous SNPs with higher level of accuracy and throughput, we hypothesize the method can be a robust approach for gene expression analysis, when adapting the rcPCR approach. The specific aims of the proposed research include: (1) Analysis of the expression level of 50 genes using SPC- SBE and rcPCR. We will first develop an expression analysis tool using SPC-SBE and rcPCR. We will use a well studied system, gene expression changes during retinal development, as a model to establish the proposed method. (2) Optimization of the sample preparation process for MALDI-TOF MS. We will enhance the accuracy of the quantitative genotyping (and thus the gene expression level measurement) by optimizing the sample crystal preparation and data analysis. (3) Verification of the proposed system for breast cancer samples. We will demonstrate our system for monitoring gene expression changes in breast cancer. The result will be compared with previous studies. The proposed method will permit high throughput, accurate and cost-effective monitoring of gene expression level changes in breast cancer. Therefore, the method will provide easy classification of breast cancer subtypes and accurate determination of progression stages, which is critical for individual patient assessment and for personalized treatment.