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
The Aurora kinases have been found to be important regulators in a number of cell cycle events including mitosis [1–3]. For these experiments, Ambion® Silencer® Select siRNAs
for human Aurora kinase A (AurkA) and Aurora kinase B (AurkB) were transfected into HeLa cells. Both siRNAs induced potent mRNA knockdown and exhibited a strong phenotype, which was pronounced on day 3 post-transfection and still evident through day 10 (Figure 1, data shown through day 6 only). The cells’ nuclei increased more than 2-fold in size and became multinuclear in appearance. Under visible light, cells transfected with siRNAs targeting AurkA and AurkB appeared larger when compared to negative control siRNA-treated cells or nontreated cells. To confirm the phenotype, cells were harvested and stained with Alexa Fluor® 594 phalloidin and mounted using VECTASHIELD® with DAPI at selected time points. The observed phenotypes were entirely consistent with an inhibition of Aurora-kinase activity . Transfection of nontargeting negative control siRNA and siRNA to an unrelated gene, the cholesterol biosynthesis enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), did not result in enlarged nuclei, providing further evidence that the observed phenotype is specific to the inhibition of Aurora kinases.
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.
As researchers use RNAi to understand gene function, analyze pathways, validate targets and develop therapeutics, it is important to optimize each key step in the experiment. In addition to validating the siRNAs and optimizing the delivery method used for a particular experimental system, it is crucial to measure RNAi effects at optimal time point(s) after transfection. In the experiment shown here, day 3 was the optimal time of harvest for phenotypic readout and it persisted through day 10. This timeline lagged behind that of maximal mRNA knockdown, which occurred on day 2.
Angie Cheng, Mu Li, Alexander (Sasha) Vlassov, Susan Magdaleno • Applied Biosystems Inc., Austin, TX