• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文会议>Conference on Ultrasensitive and Single-Molecule Detection Technologies >Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling
【24h】

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

机译：单分子荧光显微镜显微镜，用于超敏感的RNA表达分析

获取原文
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 106 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.
机译：我们开发了一种微阵列分析平台，用于微小样本的超敏感RNA表达分析。它利用新型扫描系统，用于对CM2尺寸样品上的单分子荧光检测，与专业的生物芯片组合，优化用于低自发荧光和弱的非特异性吸附。从人角蛋白细胞细胞系（HACAT）的106个细胞中提取20μg总RNA，并在Alexa647-Aha-DUTP存在下反转转录。所得标记的cDNA的1％用于复杂的杂交，以表示一组125个不同基因的定制寡核苷酸微阵列。对于低丰富的基因，可以解决与微阵列斑点杂交的单个cDNA分子。与芯片表面杂交的单个cDNA分子出现在荧光图像中的衍射限制特征。单次小波法用于定位和计数分离的cDNA信号。随后，通过不同基因的亮度分析测定局部cDNA分子的标记度。发现最多6种的变化，在传统的微阵列分析中，这将导致MRNA的相对丰度的歪曲。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号