Department of Pharmacology, The Ohio State University College of Medicine, Columbus, OH 43210, USA.
Anal Biochem. 2011 Oct 1;417(1):162-4. doi: 10.1016/j.ab.2011.06.008. Epub 2011 Jun 13.
RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets.
RNA 干扰是一种调节 mRNA 水平的细胞机制。它已被广泛用于敲低特定的蛋白质靶标。所选干扰 RNA 序列极大地影响其敲低靶标的能力。在这里，我们提出了一种构建表达针对 mRNA 不同区域的短发夹 RNA (shRNA) 的多重检测质粒的方法。在培养的细胞中进行简单的荧光测试，可方便地在 96 孔板上评估许多不同 shRNA 对 mRNA 敲低的效果。我们表明，软件预测的 shRNA 具有不同的功效，在测试的 7 个 shRNA 中只有 2 个显著敲低了它们的靶标。
