Validation of siRNA knockdowns by quantitative PCR

One day before transfection, 3,000 A549 cells or 10,000 NHBE cells per well were seeded onto 96-well plates. Transfection was performed as described above. RNA was isolated 24 hours post transfection using the MagMax 96 Total RNA isolation kit (Ambion), and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The relative amount of target mRNA was determined by singleplex quantitative PCR using pre-designed Taqman gene expression assays and Taqman Fast Advanced Master Mix (Applied Biosystems) following the manufacturer’s instructions. GAPDH was used as a reference gene, and the relative expression levels of target mRNA were normalized against cells transfected with Allstars non-targeting control siRNA. Three independent knockdown measurements were performed for each siRNA or combination of siRNAs evaluated.