Chen Stephen W, Oh Steve K W
Stem Cell Group, Bioprocessing Technology Institute, Centros, Singapore.
Methods Mol Biol. 2010;650:75-83. doi: 10.1007/978-1-60761-769-3_6.
The following protocols provide a rapid approach for establishing good working conditions for transfecting siRNAs for specific gene knockdown. By first using microscopy to evaluate efficient transfection of an inexpensive, fluorescent oligonucleotide, the researcher can later proceed with more expensive Western blot or quantitative real-time PCR (qRT-PCR) methods. Thus, the main culprit of ineffective knockdown, poor transfection, can be eliminated before engaging in tedious and time-consuming approaches for troubleshooting siRNA knockdown experiments.
以下方案提供了一种快速方法，用于建立针对特定基因敲低转染小干扰RNA（siRNA）的良好工作条件。通过首先使用显微镜评估一种廉价的荧光寡核苷酸的有效转染情况，研究人员随后可以采用更昂贵的蛋白质印迹法或定量实时聚合酶链反应（qRT-PCR）方法。因此，在采用繁琐且耗时的方法对siRNA敲低实验进行故障排除之前，可以消除敲低无效的主要原因——转染不佳。
