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

描述（由申请人提供）：在这里，我们提出了一种新的基因表达水平变化的基因组分析方法。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，并可以说明复杂疾病（如乳腺癌）在不同阶段的分子特征。因此，采用一种准确、快速和经济有效的方法对基因表达水平进行有效测量，将有助于了解乳腺癌的发展和疾病的诊断。该方法采用两种方法，固相可捕获单碱基扩展（SPC-SBE）方法和真实竞争PCR （rcPCR）方法。SPC-SBE是PI开发的一种多重基因分型方法，采用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和分子亲和系统。SPC-SBE方法包括在MS测量之前特异性分离DNA延伸片段的程序，允许遗传变异的多重基因分型的更高通量和准确性。由于SPC-SBE提供了一个强大的杂合snp基因分型，具有更高的准确性和通量，我们假设该方法可以成为基因表达分析的强大方法，当采用rcPCR方法时。本研究的具体目的包括：(1)利用SPC- SBE和rcPCR分析50个基因的表达水平。我们将首先开发一个使用SPC-SBE和rcPCR的表达分析工具。我们将使用一个经过充分研究的系统，即视网膜发育过程中基因表达的变化，作为模型来建立所提出的方法。(2)优化MALDI-TOF ms样品制备工艺，通过优化样品晶体制备和数据分析，提高定量基因分型（从而提高基因表达水平测定）的准确性。(3)对所提出的乳腺癌样本系统进行验证。我们将展示我们的监测乳腺癌基因表达变化的系统。研究结果将与以往的研究进行比较。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准确的进展阶段的确定，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.