Rapid FISH Assay for Disease-Related Nuclear RNA Transcripts

疾病相关核 RNA 转录本的快速 FISH 检测

• 批准号: 7110620
• 负责人: JOAN AURICH-COSTA
• 金额: $ 25.06万
• 依托单位: ONE CELL SYSTEMS, INC
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110620
• 关键词: biotechnology; chromosome aberrations; flow cytometry; fluorescent dye /probe; fluorescent in situ hybridization; gene expression; gene targeting; image enhancement; nucleic acid probes; nucleic acid quantitation /detection; single nucleotide polymorphism; technology /technique development

DESCRIPTION (provided by applicant): The most common diagnostic and commercial applications of FISH are in the detection of structural aberrations, such as chromosomal translocations and gene amplifications, both commonly found in a variety of cancers. This is typically accomplished using DNA probes several hundreds of kilobases (kb) in size, targeting similarly sized DNA regions. The large size of the probe constructs, primarily for increasing hybridization signal sensitivity, introduce a variety of design, labeling, and usage problems. It is also difficult to detect specific microdeletions or single-base changes using these probes. More fundamentally, these procedures target structural defects, but do not provide information on the expression status of the defect, which ultimately is involved in determining disease phenotype. To accomplish this, gene expression must be targeted, requiring detection of RNA instead of DNA. Although detection of cellular RNA using in situ hybridization has long been reported, most applications target cytoplasmic RNAs, primarily due to their increased copy number, with the intent of determining whether a specific gene is transcribed or quiescent. A novel variation of RNA detection, which greatly expands the information obtained, is detection of nuclear RNA, not cytoplasmic RNA, as the targeted molecule. Several different nuclear RNA molecules have been detected and in all cases, the nuclear RNA transcript focus is coincident with the transcribed gene. In addition, since the nuclear RNA hybridization signal was tightly restricted to a defined space, detection sensitivity was increased relative to that from the more diffuse cytoplasmic mRNA. Using a novel probe labeling method, this SBIR aims to develop a rapid, multi-color RNA FISH assay for detecting aberrant nuclear RNA transcripts, which are powerful surrogate markers for certain disease-specific genetic defects. Phase I research will use 2 disease models: Chronic Myelogenous Leukemia and Spinal Muscular Atrophy.

描述（由申请人提供）：FISH最常见的诊断和商业应用是检测结构畸变，如染色体易位和基因扩增，这两种情况通常见于各种癌症。这通常使用大小为数百个DNA酶（kb）的DNA探针来实现，靶向类似大小的DNA区域。主要用于增加杂交信号灵敏度的探针构建体的大尺寸引入了各种设计、标记和使用问题。使用这些探针也难以检测特定的微缺失或单碱基变化。更根本的是，这些程序靶向结构缺陷，但不提供关于缺陷表达状态的信息，而缺陷表达状态最终参与确定疾病表型。为了实现这一点，必须靶向基因表达，需要检测RNA而不是DNA。虽然使用原位杂交检测细胞RNA的报道由来已久，但大多数应用主要是针对细胞质RNA，主要是由于它们的拷贝数增加，目的是确定特定基因是转录还是静止。RNA检测的一种新的变化，极大地扩展了所获得的信息，是检测核RNA，而不是细胞质RNA，作为靶分子。已经检测到几种不同的核RNA分子，并且在所有情况下，核RNA转录焦点与转录的基因一致。此外，由于核RNA杂交信号被严格限制在一个确定的空间，检测灵敏度增加相对于从更弥漫的细胞质mRNA。使用一种新的探针标记方法，该SBIR旨在开发一种快速，多色RNA FISH检测异常核RNA转录本，这是某些疾病特异性遗传缺陷的强大替代标记。I期研究将使用2种疾病模型：慢性髓性白血病和脊髓性肌萎缩症。