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中文摘要
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英文摘要
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.
期刊论文(14)
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会议论文
Nonradioactive arbitrarily primed polymerase chain reaction: a novel technique for detecting genetic defects in skin tumors.
非放射性任意引发聚合酶链反应:一种检测皮肤肿瘤遗传缺陷的新技术。
DOI: 10.1111/1523-1747.ep12329949
发表时间: 1996
期刊: The Journal of investigative dermatology
影响因子: --
作者: [Vogt,T, Stolz,W, Landthaler,M, Rüschoff,J, Schlegel,J]
通讯作者: Schlegel,J
Deficiency of a novel retinoblastoma binding protein 2-homolog is a consistent feature of sporadic human melanoma skin cancer.
新型视网膜母细胞瘤结合蛋白2-同源物的缺乏是散发性人类黑色素瘤皮肤癌的一致特征。
DOI: --
发表时间: 1999
期刊: Laboratory investigation; a journal of technical methods and pathology
影响因子: --
作者: [Vogt,T, Kroiss,M, McClelland,M, Gruss,C, Becker,B, Bosserhoff,AK, Rumpler,G, Bogenrieder,T, Landthaler,M, Stolz,W]
通讯作者: Stolz,W
DOI: 10.1093/nar/gni095
发表时间: 2005-06-24
期刊: Nucleic acids research
影响因子: 14.9
作者: [Rondeau G, McClelland M, Nguyen T, Risques R, Wang Y, Judex M, Cho AH, Welsh J]
通讯作者: Welsh J
DOI: 10.1158/0008-5472.can-10-0021
发表时间: 2010-08-15
期刊: Cancer research
影响因子: 11.2
作者: [Wang Y, Xia XQ, Jia Z, Sawyers A, Yao H, Wang-Rodriquez J, Mercola D, McClelland M]
通讯作者: McClelland M
9
    Salmonella Genes Associated with Colonization of Specific Hosts
    Salmonella Genes Associated with Colonization of Specific Hosts
    Affymetrix GSC3000 microarray scanner
    • 批准号:
      6877397
    • 项目类别:
    • 资助金额:
      $13.58万
    • 财政年份:
      2005
    • 负责人:
      MICHAEL MCCLELLAND
    • 依托单位:
    AFFYMETRIX GSC3000 MICROARRAY SCANNER: CANCER
    • 批准号:
      7166552
    • 项目类别:
    • 资助金额:
      $11.27万
    • 财政年份:
      2005
    • 负责人:
      MICHAEL MCCLELLAND
    • 依托单位:
    海外基金