siRNA-Induced mRNA Knockdown and Phenotype: How to Measure and When to Measure
|A typical RNAi workflow consists of transfecting siRNAs into a desired cell line, measuring target mRNA knockdown, and observing the resulting phenotype. Optimization of each step is critical to the success of the experiment, and should include a determination of appropriate time points for observation of both mRNA knockdown and the appearance of the resulting phenotypic response. There may be a delay between the two. Optimal timing can be influenced by factors such as the gene target, siRNAs used, cell line, and specific phenotypic assays. In this report, Applied Biosystems scientists investigated the duration of an expected phenotype in parallel with measuring mRNA knockdown at various time points after siRNA transfection.|
Optimize Timing of the Phenotypic Assay After Transfection
Figure 1. mRNA Knockdown and Phenotypic Changes Induced by siRNAs Targeting AurkA and AurkB. HeLa cells were transfected with 5 nM siRNAs targeting AurkA or AurkB in duplicate. In one set, cells were stained with Alexa Fluor® 594 phalloidin (red, stains actin) and mounted using VECTASHIELD® with DAPI (blue, stains nuclei). In another set, real-time RT-PCR was performed using the TaqMan® Gene Expression Cells-to-CT™ Kit. Knockdown data were calculated relative to cells transfected with Ambion® Silencer® Select Negative Control #1 siRNA.
In contrast to the timing of phenotypic changes, maximal mRNA knockdown was observed on day 2 post-transfection. This is typical for siRNA experiments. Maximal mRNA knockdown is often seen 24–48 hours after transfection, while maximal protein and phenotypic responses typically require 48–96 hours. This lag in phenotypic response is due to the fact that potent siRNAs induce a rapid reduction in target mRNA levels and protein synthesis, but do not affect the quantity or turnover rate of protein.
Angie Cheng, Mu Li, Alexander (Sasha) Vlassov, Susan Magdaleno • Applied Biosystems Inc., Austin, TX