Holmes Katherine, Williams Catrin M, Chapman Elinor A, Cross Michael J
Department of Pharmacology and Therapeutics, School of Biomedical Sciences, University of Liverpool, Liverpool, L69 3GE, UK.
BMC Res Notes. 2010 Mar 3;3:53. doi: 10.1186/1756-0500-3-53.
RNA interference (RNAi) has been one of the most rapidly expanding areas of biological research in the past decade, revolutionizing the ability to analyze gene function. Thorough validation of siRNA duplexes is required prior to use in experimental systems, ideally by western blotting to show a reduction in protein levels. However, in many cases good antibodies are not available, and researchers must rely on RT-qPCR to detect knockdown of the mRNA species.
We have observed a phenomenon that gives a disparity between analyzing small interfering RNA (siRNA) efficacy by western blotting of the protein levels and real-time quantitative PCR (RT-qPCR) measurement of mRNA levels. Detection of this phenomenon was dependent upon the location of the target amplicon for PCR primers within the mRNA.
Our data suggests that for certain mRNAs, degradation of the 3' mRNA fragment resulting from siRNA mediated cleavage is blocked, leaving an mRNA fragment that can act as a template for cDNA synthesis, giving rise to false negative results and the rejection of a valid siRNA duplex. We show that this phenomenon may be avoided by the careful design of RT-qPCR primers for each individual siRNA experiment.
在过去十年中，RNA干扰（RNAi）一直是生物研究领域中发展最为迅速的领域之一，它彻底改变了分析基因功能的能力。在将小干扰RNA（siRNA）双链体用于实验系统之前，需要对其进行全面验证，理想情况下是通过蛋白质印迹法来显示蛋白质水平的降低。然而，在许多情况下，没有合适的抗体，研究人员必须依靠逆转录定量聚合酶链反应（RT-qPCR）来检测mRNA种类的敲低情况。
我们观察到一种现象，即通过蛋白质水平的蛋白质印迹法分析小干扰RNA（siRNA）的功效与通过实时定量PCR（RT-qPCR）测量mRNA水平之间存在差异。这种现象的检测取决于PCR引物的目标扩增子在mRNA中的位置。
我们的数据表明，对于某些mRNA，由siRNA介导的切割导致的3'mRNA片段的降解被阻断，留下一个可以作为cDNA合成模板的mRNA片段，从而产生假阴性结果并导致有效的siRNA双链体被否定。我们表明，通过为每个单独的siRNA实验精心设计RT-qPCR引物，可以避免这种现象。
