MESSENGER RNA PROFILING BY SINGLE MOLECULE COUNTING

具有精确测量肿瘤细胞中基因表达水平的能力是很重要的。现有的技术允许在含有至少10,000个细胞的样品中评估基因表达水平。因此，需要一种方法，允许在含有较少肿瘤细胞的样品中进行这种测量。该建议的一个具体目标是补充一种新方法，该方法将产生含有少于200个细胞的组织样品中特定mRNA分子相对浓度的定量数据。序列检测是在寡核苷酸微阵列上完成的，使用靶向DNA连接步骤与滚动环扩增（RCA）单分子检测系统相结合。每个目标检测事件都会产生一个引物，可以被RCA放大。RCA产生的每个扩增DNA分子都定位在阵列表面，并成像为离散的荧光信号，表明特定的分子连接事件。表达谱生成为单分子计数的直方图。此外，DNA连接步骤可以适用于检测含有点突变的mrna。这种能力将用于在1000个野生型mRNA分子的背景下检测一个体细胞突变mRNA分子，使用K- ras突变作为实验模型。用于分析100种不同基因产物的阵列将包括已知在癌细胞中上调或下调的mrna，野生型或突变K-ra mrna，以及管家基因。该系统将纳入适当的控制措施，以确保其可靠性。另一个具体的目的是评估这种高灵敏度检测技术与激光辅助组织显微解剖的耦合。我们将结合这两种技术来展示高分辨率组织分析的能力：(a)正常和癌前列腺，以及不同程度的前列腺上皮内瘤变的前列腺组织；(b)正常的结肠上皮，以及不同程度不典型增生的腺瘤上皮和结肠腺癌。在含有10-20个细胞的样品中测量基因表达水平的能力，以及在几个已知高频改变的位点上检测体细胞点突变的能力，将为解决在发育不良和癌症的小细胞群中基因表达谱可能存在的微异质性问题提供基础设施。

It is important to have the capability to perform precise measurements of gene expression levels in tumor cells. Available technology permits the assessment of levels of gene expression in samples containing a minimum of 10,000 cells. Thus, there is a need for methods that will permit such measurements in samples containing fewer tumor cells. A specific aim of this proposal is to complement a new approach that will yield quantitative data on the relative concentration of specific mRNA molecules in tissue samples containing less than 200 cells. Sequence detection is accomplished on oligonucleotide microarrays, using a target-directed DNA ligation step coupled to a Rolling Circle Amplification (RCA) unimolecular detection system. Each target detection event generates a primer that can be amplified by RCA. Each amplified DNA molecule generated by RCA remains localized on the array surface, and is imaged as a discrete fluorescent signal, indicative of a specific molecular ligation event. Expression profiles are generated as histograms of single molecule counts. Additionally, the DNA ligation step can e adapted to the detection of mRNAs containing point mutations. This capability will be developed for detection of one somatic mutant mRNA molecule in a background of 1000 wild type mRNA molecules, using K- ras mutations as an experimental model. Arrays for the analysis of 100 different gene products will include mRNAs known to be up- or down- regulated in cancer cells, wild type or mutant K-ra mRNAs, and housekeeping genes. Adequate controls will be incorporated in the system to insure its reliability. Another specific aim is to evaluate the coupling of this highly sensitive detection technology with laser- assisted tissue microdissection. We will combine these two technologies to demonstrate the capability for high resolution tissue analysis in: (a) normal and cancerous prostate, and prostate tissue with varying degrees of prostatic intraepithelial neoplasia; (b) normal colonic epithelium, as well as adenomatous epithelium with varying degrees of dysplasia, and colonic adenocarcinoma. The capability for measuring gene expression levels in samples containing as few as 10-20 cells, together with the capability for detection of somatic point mutations at several loci known to be altered with high frequency, will provide the infrastructure to address the question of possible microheterogeneity in gene expression profiles in small clusters of cells in dysplasia and cancer.