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.