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重要的。现有技术允许评估含有至少10，000个细胞的样品中的基因表达水平。因此，需要允许在含有较少肿瘤细胞的样品中进行这种测量的方法。该提案的一个具体目的是补充一种新的方法，该方法将产生关于含有少于200个细胞的组织样本中特定mRNA分子的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连接步骤与滚环扩增（RCA）单分子检测系统偶联。每个靶检测事件产生可通过RCA扩增的引物。由RCA产生的每个扩增的DNA分子保持定位在阵列表面上，并且成像为离散的荧光信号，指示特定的分子连接事件。表达谱生成为单分子计数的直方图。另外，DNA连接步骤可适于检测含有点突变的mRNA。这种能力将被开发用于在1000个野生型mRNA分子的背景中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、野生型或突变型K-ra mRNA和管家基因。该系统将纳入适当的控制措施，以确保其可靠性。另一个具体目标是评估这种高灵敏度检测技术与激光辅助组织显微切割的耦合。我们将联合收割机结合这两种技术，以证明在以下方面进行高分辨率组织分析的能力：（a）正常和癌性前列腺，以及具有不同程度前列腺上皮内瘤形成的前列腺组织;（B）正常结肠上皮，以及具有不同程度异型增生的腺瘤上皮和结肠腺癌。在含有少至10-20个细胞的样品中测量基因表达水平的能力，以及在已知以高频率改变的几个基因座处检测体细胞点突变的能力，将提供基础设施来解决在异型增生和癌症的小细胞簇中基因表达谱中可能的微观异质性的问题。

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.