Quantification of miRNA abundance in single cells using locked nucleic acid-FISH and enzyme-labeled fluorescence.
Quantification of miRNA abundance in single cells using locked nucleic acid-FISH and enzyme-labeled fluorescence.
复制标题
使用锁核酸-FISH 和酶标记荧光定量单细胞中的 miRNA 丰度。
DOI：
10.1007/978-1-60761-901-7_5
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Tsourkas,Andrew
中科院分区：
文献类型：
--
作者：
Lu,Jing;Tsourkas,Andrew
在单细胞水平上定量miRNA丰度并对其空间分布进行成像的能力可能会导致对miRNA和miRNA相关基因调控网络的生物学作用的独特见解。该方案描述了使用荧光原位杂交（FISH）对单细胞中的miRNA进行定量成像的方法。该方法结合了锁核酸（LNA）独特的miRNA识别特性和称为酶标记荧光（ELF）的信号放大技术。虽然这两种方法先前已被证明可以增加FISH中的检测特异性和/或灵敏度，但将这些技术组合到一个方案中允许单分子检测。具体而言，单个miRNA被鉴定为明亮的光稳定荧光点。发现动态范围跨越三个数量级，并且每个细胞的平均miRNA拷贝数在通过定量RT-PCR获得的测量值的17.5%内。
The ability to quantify miRNA abundance at the single-cell level and image its spatial distribution could lead to unique insight into the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for quantitatively imaging miRNAs in single cells using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA) with the signal amplification technology known as enzyme-labeled fluorescence (ELF). Although both approaches have previously been shown to increase detection specificity and/or sensitivity in FISH, combining these techniques into one protocol allows for single molecule detection. Specifically, individual miRNAs are identified as bright, photostable fluorescent spots. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.
DOI：
10.1016/s0021-9258(19)75701-9
发表时间：
1987-02
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
M. Inui;A. Saito;S. Fleischer
通讯作者：
M. Inui;A. Saito;S. Fleischer
DOI：
--
发表时间：
1975
期刊：
Biochimica et Biophysica Acta
影响因子：
--
作者：
Gerhard Meissner
通讯作者：
Gerhard Meissner