Arrays and Targets for Nascent Transcripts

描述(由申请人提供)： 在微阵列实验中，增加种群中更稀有RNA的检测的一种方法是使用只标记一小部分RNA的方法。这些低复杂性表示(LCR)提高了对所选RNA的检测的灵敏度，并且还可以降低由目标复杂性造成的背景的任何部分。我们使用带有随机引物的RNA的聚合酶链式反应(RAP)来丰富RNA种群的亚群。该方法是根据与引物的偶然性匹配来选择RNA中的内部扩增片段，具有简单、稳健的特点。我们已经证明，与传统玻片阵列或Affymetrix阵列上的总RNA相比，LCR可以将mRNA的检测下限提高一个数量级，同时保持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，旨在最大化RAP LCR的覆盖范围。这将允许检测到罕见的mRNAs。(2)构建LCRs的“内含子”阵列，由于其敏感性增加，将允许测量新生转录本(HnRNAs)的水平。当与适当的阵列杂交时，每个LCR可能能够检测到细胞中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，到目前为止，这些方法主要集中在细胞中成熟mRNA的丰度上。(3)通过对RNA群体的限制性片段亚集进行聚合酶链式反应来产生LCR。(4)采用线性扩增策略，从hnRNA中与分散重复序列相邻的序列进行LCR。该LCR将与适当的定制数组混合。四个目标的结果将进行比较。然后，在Aim(5)中，最佳LCR策略和相应的优化阵列将被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mRNAs。新调控的发现可以整合到这个具有良好特征的系统中，以前已知的调控基因可以通过比较初始转录和稳定状态RNA水平，根据其调控的转录和转录后成分进行解析。

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.