RNA interference, the biological mechanism by which double-stranded RNA induces gene silencing through targeting complementary mRNA for degradation, is revolutionizing the way researchers study gene function. An efficient way to validate gene knockdown in RNA interference experiments is to use highly effective siRNAs and to monitor the resultant decrease in mRNA levels using Real-Time PCR. This article illustrates the combined use of Ambion’s Silencer® siRNAs and Applied Biosystems TaqMan® Gene Expression Assays to generate high quality, accurate, and reproducible data.
Fast and Accurate Gene Silencing
HeLa cells were transfected with Silencer
siRNAs targeting 52 kinase transcripts, and mRNA knockdown levels remaining after treatment were measured using TaqMan Gene Expression Assays. Data in Figure 1 indicate that 71% of the transcripts resulted in 80% or more mRNA knockdown levels and overall >92% of siRNAs produced significant levels of transcript reduction. Standard deviation between replicated experiments was within 10% of knockdown. The location of the TaqMan Gene Expression Assay probe relative to the location of the siRNA did not significantly influence the measurement of knockdown.
Figure 1. Silencer
® siRNAs Induced mRNA Knockdown with >80% Efficiency as Measured by TaqMan® Gene Expression Assays. HeLa cells were transfected in 96-well plates with siRNAs (100 nM) targeting 52 human transcripts from the Silencer
Kinase siRNA Library. 48 hrs post-transfection, total RNA was isolated and converted to cDNA. Two positive control siRNAs and a non-targeting negative control siRNA were included. Three siRNAs designed to the same transcript were all tested. Each siRNA was transfected in triplicate. Levels of mRNA remaining after siRNA treatment were assessed using Real-Time PCR with TaqMan® Gene Expression Assays. Additionally, detection of 18S rRNA was used to normalize total RNA purified from all samples.
A Complete Solution
Ambion, an Applied Biosystems Business, and Applied Biosystems have combined their product lines to provide a complete solution for performing gene silencing experiments and for validating the results. Ambion’s Silencer Pre-designed siRNAs (and libraries) that target >98% of all human, mouse, and rat genes in the NCBI RefSeq database eliminate costly and time-consuming siRNA validation (siRNAs to genes of other genomes also available). Applied Biosystems offers TaqMan Gene Expression Assays for >785,000 genes in human, mouse, rat, and other genomes. These assays can be used with the TaqMan Universal PCR Master Mix and Applied Biosystems 7900HT Fast Real-Time PCR System for a complete validation solution.