KTC--AN ACCURATE METHOD FOR TRANSCRIPTOME ANALYSIS

描述：（申请人的摘要）我们提出了基于PCR的 用于准确和可重复的转录水平测定的方法， 复杂的混合物 该方法，kRT-PCR（动力学监测的反向PCR）， 转录启动的PCR），支持全自动定量 使用总细胞RNA作为模板的转录本丰度， 转录物特异性引物对。 定量来自单个 使用单一的热稳定DNA聚合酶进行反应。 计算机控制的 数码相机监测PCR产物积累的循环动力学 通过荧光。 数字图像分析提供了完整的计算机数据 装卸. 从PCR动力学计算每个细胞的转录本拷贝数 产物积累（与细胞中的转录本丰度直接相关） 细胞RNA模板）。 初步结果表明，kRT-PCR检测 定量两种生理或生理条件之间的转录水平差异， 遗传状态在20%的因子内。 定量绝对mRNA水平 通过kRT-PCR测定，在2倍内。 实验被提议为 通过以下方法进一步提高kRT-PCR转录物测定的准确性 改进酶学。 初步结果表明，kRT-PCR检测很容易 定量酵母转录物，范围为每个细胞595至0.03个拷贝。 我们 建议进一步评估该检测试剂盒的检测限。 方法是 建议通过以下方式增加kRT-PCR转录本测定的通量： 另一个数量级来定量酵母基因组当量， 分析. 实验计划包括许多不同的应用程序 用于kRT-PCR测定，包括：1. 鉴别和定量 由已知的和计算注释的ORF编码的罕见转录物。 2. 由单个基因编码的多个转录物的定量作为一种基因表达。 差异RNA剪接和/或多个启动子元件的结果。 3. mRNA半衰期的测定。 4. 基因解剖 转录路径和调控网络。 5. 基因拷贝测定 完整细胞基因组的数量。 6. 后生动物定量 使用已知EST特异性引物对的转录本。 kRT-PCR检测 精度高，检测范围宽，并联灵活 在多种生理或遗传学中的多种转录物的定量 国家以及细胞类型。 这些特征使得该测定能够解决 广泛的基于基因组学的分析，包括但不限于：1. 低丰度转录物的定量。 2. 分析了一个协调的 在多种细胞类型中表达的、调节的转录物子集，或 基因状态 3. 细胞类型特异性转录标志物的定量 在异质细胞群（例如器官或组织）中， 细胞因子或治疗药物治疗。

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.