Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohrgasse 9/3, Vienna, Austria.
Gregor Mendel Institute, Dr. Bohrgasse 3, Vienna, Austria.
Plant Methods. 2014 May 31;10:15. doi: 10.1186/1746-4811-10-15. eCollection 2014.
A multitude of different imaging systems are already available to image genetically altered RNA species; however, only a few of these techniques are actually suitable to visualize endogenous RNA. One possibility is to use fluorescently-labelled and hybridization-sensitive probes. In order to yield more information about the exact localization and movement of a single RNA molecule, it is necessary to image such probes with highly sensitive microscope setups. More challenges arise if such experiments are conducted in plant cells due to their high autofluorescence and demanding transfection procedures.
Here, we report in planta imaging of single RNA molecules using fluorescently labeled molecular beacons. We tested three different transfection protocols in order to identify optimal conditions for transfection of fluorescent DNA probes and their subsequent detection at the single molecule level.
We found that an optimized heat shock protocol provided a vastly improved transfection method for small DNA molecules which were used for subsequent single RNA molecule detection in living plant suspension cells.
目前已有多种不同的成像系统可用于对遗传修饰的 RNA 进行成像；然而，这些技术中只有少数真正适用于对内源性 RNA 进行可视化。一种可能性是使用荧光标记和杂交敏感探针。为了获取有关单个 RNA 分子的确切定位和运动的更多信息，有必要使用高灵敏度的显微镜设置来对这些探针进行成像。如果在植物细胞中进行此类实验，则会面临更多挑战，因为植物细胞的自发荧光水平较高，并且转染程序要求较高。
在这里，我们使用荧光标记的分子信标报告了植物体内单个 RNA 分子的成像。我们测试了三种不同的转染方案，以确定最佳条件，以用于转染荧光 DNA 探针及其随后在单细胞水平上的检测。
我们发现，优化的热休克方案为小 DNA 分子的转染提供了一种大大改进的方法，这些小 DNA 分子随后用于在活的植物悬浮细胞中检测单个 RNA 分子。
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