Taqman fast advanced 2 master mix

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.