Multiplexed optical coding nanobeads and their application in single-molecule counting analysis for multiple gene expression analysis
Multiplexed optical coding nanobeads and their application in single-molecule counting analysis for multiple gene expression analysis
复制标题
多重光学编码纳米珠及其在多基因表达分析的单分子计数分析中的应用
DOI：
10.1016/j.aca.2015.05.042
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发表时间：
2015
影响因子：
6.2
通讯作者：
Jin Wenrui
中科院分区：
文献类型：
--
作者：
Li Lu;Shen Liping;Zhang Xiaoqian;Shui Lingling;Sui Benhui;Zhang Xiaoli;Zhao Xiaofan;Jin Wenrui
通过将三种不同染料（Cy5、FAM和AMCA）标记的编码DNA以精确控制的比例与生物素化的报告DNA杂交，开发了一种用于制备多重光学编码纳米珠（MOCNBs）的方法，所述报告DNA修饰为直径为300 nm的磁性链霉亲和素包被的纳米珠。MOCNB的颜色可以通过重叠对应于Cy5（红色）、FAM（绿色）和AMCA（蓝色）的发射的MOCNB的三个单原色荧光图像来观察。MOCNB可以在常规荧光显微镜下容易地鉴定。不同颜色的MOCNB可作为单分子计数分析（SMCA）的多重光学编码标记，并可用于多基因表达分析（MGEA）。在基于SMCA的MGEA技术中，细胞中的多个信使RNA（mRNA）可以通过它们的互补DNA（cDNA）通过计数与用MOCNB标记的单个cDNA分子对应的具有相同颜色的亮点来同时定量。我们测量了鳞翅目昆虫Helicoverpa armigerain 100 HaEpi细胞中三个基因在类固醇激素诱导和不诱导的情况下的表达谱，以证明使用MOCNBs的SMCA为基础的MGEA技术。
A method for fabrication of multiplexed optical coding nanobeads (MOCNBs) was developed by hybridizing three types of coding DNAs labeled with different dyes (Cy5, FAM and AMCA) at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated nanobeads with a diameter of 300 nm. The color of the MOCNBs could be observed by overlapping three single-primary-color fluorescence images of the MOCNBs corresponding to emission of Cy5 (red), FAM (green) and AMCA (blue). The MOCNBs could be easily identified under a conventional fluorescence microscope. The MOCNBs with different colors could serve as the multiplexed optical coding labels for single-molecule counting analysis (SMCA) and be used in multi-gene expression analysis (MGEA). In the SMCA-based MGEA technique, multiple messenger RNAs (mRNAs) in cells could be simultaneously quantified through their complementary DNAs (cDNAs) by counting the bright dots with the same color corresponding to the single cDNA molecules labeled with the MOCNBs. We measured expression profiles of three genes from Lepidoptera insectHelicoverpa armigerain ∼100 HaEpi cells with and without steroid hormone inductions to demonstrate the SMCA-based MGEA technique using MOCNBs.