Taqman gene expression assay primer probes

Centyrins were assessed for their ability to mediate uptake of siRNAs into various cell lines in the absence of transfection reagent. Cells were plated in 96-well plates (5,000–10,000 cells per well) for 24 h and then treated with Centyrin-siRNA conjugates in duplicate for 72 h. Cells were lysed with Protein Quant Sample Lysis Reagent (Applied Biosystems, Waltham, MA, USA). Lysates were frozen at −80°C until analysis. Samples were thawed on ice, and RNA in cell lysate was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in a ProFlex ThermoCycler (Thermo Scientific, Waltham, MA, USA) according to manufacturer’s instructions. qPCR of cDNA was performed using TaqMan Fast Advanced 2× Master Mix (Applied Biosystems) with TaqMan Gene Expression Assay Primer/Probes (Applied Biosystems) for CTNNb1 and peptidyl-prolyl cis-trans isomerase B (PPIB) on a ViiA 7 Real-Time PCR System (Applied Biosystems). Target gene CT values were normalized by subtracting the PPIB CT value to obtain a deltaCT. The average of untreated deltaCT values was then subtracted from the sample deltaCT. Relative mRNA level was then determined using the equation, % Gene expression = 100 × 2^{(−deltadeltaCT)}. Data were log transformed on the x axis, then analyzed using nonlinear regression, applying a 3-parameter model to determine IC₅₀.