Arrays and Targets for Nascent Transcripts

新生转录本的阵列和靶标

• 批准号: 7245019
• 负责人: MICHAEL MCCLELLAND
• 金额: $ 36.6万
• 依托单位: SIDNEY KIMMEL CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7245019
• 关键词: Biological; Cell Cycle; Cells; Custom; Data; Detection; Effectiveness; Exons; Genes; Genetic Transcription; Glass; Hela Cells; Heterogeneous Nuclear RNA; Human Cell Line; Introns; Label; Ligation; Mammalian Cell; Measurement; Measures; Messenger RNA; Metabolism; Methods; Modeling; Monitor; Nested PCR; Nucleic Acids; Numbers; Oligonucleotides; Performance; Polymerase Chain Reaction; Population; Process; RNA; RNA Sequences; RNA Splicing; RNA amplification; Regulation; Sampling; Site; Slide; Surveys; System; Testing; Thinking; Transcript; Update; base; cost; design; improved; mRNA Decay; research study

DESCRIPTION (provided by applicant): In microarray experiments, one way to increase the detection of rarer RNAs in a population is to use methods that label small portions of only some of the RNAs. These "low complexity representations" (LCRs) increase the sensitivity of detection of the RNAs selected and may also reduce any part of the background contributed by target complexity. We have used PCR of RNA with arbitrary primers (RAP) to enrich for subsets of RNA populations. The method selects internal amplicons in RNAs based on chance matches with the primers, and is simple and robust. We have demonstrated that LCRs can improve mRNA detection limits by an order of magnitude compared to total RNA on conventional glass slide arrays or Affymetrix arrays, while preserving the ratio of expression differences between two samples. We propose to expand the utility of LCR methods in four aims. (1) Build cheap custom arrays that are designed to maximize coverage by RAP LCRs. This will allow rare mRNAs to be detected. (2) Construct "intron" arrays for LCRs that, due to their increased sensitivity, will allow measurement of the level of nascent transcripts (hnRNAs). When hybridized to an appropriate array, each LCR may be able to detect 30% or more of the nascent transcripts in the cell, including some of the rarest. This will improve upon current methods for monitoring RNAs that, until now, have primarily focused on the abundance of the mature mRNA in the cell. (3) Generate LCRs by PCR of restriction fragment subsets of the RNA population. (4) A linear amplification strategy will be applied to make an LCR from sequences adjacent to dispersed repeats in hnRNA. This LCR will be hybridized to the appropriate custom array. The results of the four aims will be compared. Then, in Aim (5) the best LCR strategy, and the corresponding optimized array, will be applied to study nascent transcription and rare mRNAs in the cell cycle in human cell line models. Discoveries of new regulation can be integrated into this well characterized system, and previously known regulated genes can be parsed according to transcriptional vs. post-transcriptional components of their regulation by comparing nascent transcription to steady state RNA levels.

描述（由申请人提供）： 在微阵列实验中，增加对群体中较稀有 RNA 的检测的一种方法是使用仅标记某些 RNA 的一小部分的方法。这些“低复杂性表示”(LCR) 提高了所选 RNA 检测的灵敏度，并且还可以减少由目标复杂性造成的背景的任何部分。我们使用任意引物 (RAP) 对 RNA 进行 PCR 来富集 RNA 群体的子集。该方法根据与引物的机会匹配来选择 RNA 中的内部扩增子，并且简单且可靠。我们已经证明，与传统载玻片阵列或 Affymetrix 阵列上的总 RNA 相比，LCR 可以将 mRNA 检测限提高一个数量级，同时保留两个样本之间的表达差异比率。我们建议在四个目标上扩展 LCR 方法的实用性。 (1) 构建廉价的定制阵列，旨在最大限度地提高 RAP LCR 的覆盖范围。这将允许检测到稀有的 mRNA。 (2) 构建 LCR 的“内含子”阵列，由于其敏感性增加，可以测量新生转录物 (hnRNA) 的水平。当与适当的阵列杂交时，每个 LCR 可能能够检测细胞中 30% 或更多的新生转录本，包括一些最稀有的转录本。这将改进目前监测 RNA 的方法，迄今为止，这些方法主要关注细胞中成熟 mRNA 的丰度。 (3) 通过 RNA 群体的限制性片段子集的 PCR 生成 LCR。 (4) 将应用线性扩增策略从与 hnRNA 中分散的重复序列相邻的序列制备 LCR。该 LCR 将与适当的定制阵列杂交。将比较四个目标的结果。然后，在目标（5）中，最佳LCR策略和相应的优化阵列将应用于研究人类细胞系模型中细胞周期中的新生转录和稀有mRNA。新调控的发现可以整合到这个明确表征的系统中，并且可以通过比较新生转录与稳态 RNA 水平，根据其调控的转录与转录后成分来解析先前已知的调控基因。