Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.
Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.
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DOI：
10.1002/smll.201401137
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发表时间：
2015-05
期刊：
影响因子：
--
通讯作者：
Espinosa HD
中科院分区：
文献类型：
--
作者：
Giraldo-Vela JP;Kang W;McNaughton RL;Zhang X;Wile BM;Tsourkas A;Bao G;Espinosa HD
单细胞分析的新技术使基因表达和系统生物学的新发现成为可能。对单个细胞的时间依赖性测量是必要的，然而目前使用的常见单细胞分析技术需要裂解细胞、悬浮细胞或长的转染孵育时间，从而干扰随时间跟踪单个细胞的能力。在这里，提出了一种使用分子信标检测活单细胞中mRNA表达的方法，所述分子信标通过纳米峰探针电穿孔（NFP-E）转染到单细胞中。分子信标是寡核苷酸，其在与mRNA靶结合时发射荧光，使其可用于活细胞的空间和时间研究。NFP-E用于检测基于DNA的信标和基于RNA的信标，所述基于DNA的信标检测甘油醛3-磷酸脱氢酶，所述基于RNA的信标检测克隆在绿色荧光蛋白mRNA中的序列。结果表明，转染和mRNA检测的成像分析可以在电穿孔后数秒内进行，而不会干扰粘附的细胞。此外，它表明，时间依赖性检测的mRNA表达是可行的，通过在不同的时间点重复相同的单细胞。这种技术将是特别有用的细胞分化的研究，其中mRNA表达的几个测量需要随着时间的推移。
New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.
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影响因子：
14.9
作者：
Chen AK;Behlke MA;Tsourkas A
通讯作者：
Tsourkas A
影响因子：
10.8
作者：
Kang W;Yavari F;Minary-Jolandan M;Giraldo-Vela JP;Safi A;McNaughton RL;Parpoil V;Espinosa HD
通讯作者：
Espinosa HD
影响因子：
7.4
作者：
Medley, CD;Drake, TJ;Tan, WH
通讯作者：
Tan, WH
影响因子：
12.4
作者：
Gasiorowski, JZ;Dean, DA
通讯作者：
Dean, DA
影响因子：
56.9
作者：
Femino, A;Fay, FS;Singer, RH
通讯作者：
Singer, RH