KTC--AN ACCURATE METHOD FOR TRANSCRIPTOME ANALYSIS

描述：(申请人摘要)我们提出了基于聚合酶链式反应的应用 一种准确和可重复性的基因转录水平测定方法 复杂的混合物。这种方法，KRT-PCR(动态监测反向 转录启动的聚合酶链式反应)，支持全自动定量 以细胞总RNA为模板的转录本丰度 转录特异的引物对。量化是从单个 使用单一的耐热DNA聚合酶进行反应。一台计算机控制的 数码相机监测聚合酶链式反应产物积累的循环动力学 通过荧光检测。数字图像分析提供完整的计算机数据 正在处理。根据聚合酶链式反应动力学计算每个细胞的转录本拷贝数 产物积累(与转录本丰度直接相关) 细胞RNA模板)。初步结果表明，KRT-PCR检测 量化两个生理性或非生理性之间的转录水平差异 遗传状态在20%的范围内。绝对信使核糖核酸水平是定量的 KRT-PCR法检测两个因素内。建议进行实验，以 进一步提高KRT-PCR转录本测定的准确性 改进了酶学。初步结果表明，krt-pcr分析很容易。 定量酵母转录本，每个细胞从595到0.03个拷贝。我们 建议进一步评估该检测方法的检测限。方法是 建议通过以下方式增加KRT-PCR转录本测定的吞吐量 量化酵母基因组当量的另一个数量级 分析。实验计划包括一些不同的应用 用于KRT-PCR检测，包括：1.鉴定和定量 由已知和计算注释的ORF编码的罕见转录本。2. 单个基因编码的多个转录本的定量 差异RNA剪接和/或多个启动子元件的结果。 3.信使核糖核酸半衰期测定4.青枯病遗传解剖 转录途径和调控网络。5.基因拷贝的测定 在完整的细胞基因组中。6.后生动物的定量 使用已知EST特有的引物对转录。KRT-PCR法 具有精度高、检测范围广、并联灵活等特点 多个生理或基因中的多个转录本的定量 状态以及细胞类型。这些功能使该分析能够解决 广泛的基于基因组学的分析，包括但不限于：1. 低丰度转录本的定量。2.对一个协调问题的分析 在各种各样的细胞类型中表达的、受调控的转录子集 基因状态。3.细胞类型特异性转录本标记的定量 在异质细胞群(例如器官或组织)中 细胞因子或治疗性药物治疗。

DESCRIPTION: (Applicant's abstract) We propose applications of a PCR-based method for accurate and reproducible transcript level determinations in complex mixtures. This method, kRT-PCR (kinetically-monitored reverse transcription-initiated PCR), supports fully automated quantitation of transcript abundance using total cellular RNA as template and transcript-specific primer pairs. Quantitation derives from a single reaction using a single thermostable DNA polymerase. A computer-controlled digital camera monitors the cyclewise kinetics of PCR product accumulation by fluorescence. Digital image analysis provides for full computer data handling. Transcript copies per cell are computed from the kinetics of PCR product accumulation (directly related to transcript abundance in the cellular RNA template). Preliminary results show that the kRT-PCR assay quantitates transcript level differences between two physiological or genetic states within a factor of 20%. Absolute mRNA levels are quantitated by kRT-PCR assay within a factor of two. Experiments are proposed to further refine the accuracy of kRT-PCR transcript determinations through improved enzymology. Preliminary results show that kRT-PCR assays readily quantitate yeast transcripts ranging from 595 to 0.03 copies per cell. We propose to further assess this assay's detection limits. Approaches are proposed to increase throughput of kRT-PCR transcript determinations by another order of magnitude to quantitate a yeast genome equivalent per analysis. The Experimental Plan includes a number of different applications for the kRT-PCR assay, including: 1. Identification and Quantitation of rare transcripts encoded by known and computationally annotated ORFs. 2. Quantitation of multiple transcripts encoded by a single gene as a consequence of differential RNA splicing and/or multiple promoter elements. 3. Determination of mRNA half lives. 4. Genetic dissection of transcription paths and regulatory networks. 5. Determination of gene copy number in complete cellular genomes. 6. Quantitation of metazoan transcripts using primer pairs specific for known ESTs. The kRT-PCR assay has high accuracy, wide detection range, and flexibility for parallel Quantitation of multiple transcripts in multiple physiological or genetic states as well as cell types. These features enable this assay to address a wide range of genomics-based analyses including, but are not limited to: 1. Quantitation of low abundance transcripts. 2. Analyses of a coordinately expressed, regulated subset of transcripts in a wide varied of cell types or genetic states. 3. Quantitation of cell type specific transcript markers in heterogeneous cell populations (e.g. organs or tissues) as a function of cytokine or therapeutic drug treatment.