Arrays and Targets for Nascent Transcripts

描述（由申请人提供）： 在微阵列实验中，增加群体中稀有RNA检测的一种方法是使用仅标记某些RNA的小部分的方法。这些“低复杂性表示”（LCR）增加了所选RNA的检测灵敏度，并且还可以减少由靶复杂性贡献的背景的任何部分。我们已经使用PCR的RNA与任意引物（RAP），以丰富的子集的RNA群体。该方法基于与引物的偶然匹配来选择RNA中的内部扩增子，并且简单且稳健。我们已经证明，与常规载玻片阵列或Affytron阵列上的总RNA相比，LCR可以将mRNA检测限提高一个数量级，同时保持两个样品之间的表达差异比率。我们建议扩大LCR方法的效用在四个目标。(1)构建廉价的定制阵列，旨在最大限度地提高RAP LCR的覆盖率。这将允许检测到罕见的mRNA。(2)构建LCR的“内含子”阵列，由于其灵敏度增加，将允许测量新生转录物（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%或更多的新生转录物，包括一些最稀有的转录物。这将改善目前用于监测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RNA群体的限制性片段子集的PCR产生LCR。(4)将应用线性扩增策略从hnRNA中与分散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。四个目标的结果将进行比较。然后，在目标（5）中，最佳LCR策略和相应的优化阵列将被应用于在人类细胞系模型中研究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充分表征的系统中，并且通过比较新生转录与稳态RNA水平，可以根据其调控的转录与转录后组分来解析先前已知的调控基因。

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.