Quantitative analysis by real-time RT-PCR

RNA was isolated from the MDA-MB-157 and APC shRNA1 cells using Tri Reagent (Molecular Research Center, Cincinnati, OH), and cDNA synthesis was performed with iScript (Bio-Rad Laboratories, Hercules, CA). Real-time RT-PCR was performed with Power SYBR Green master mix (Applied Biosystems, Foster City, CA) and 50ng cDNA. Primer sequences are presented in Table 2. The amplification program included: 2 initial steps at 50°C for 2 minutes and 95°C for 10 minutes; 40 cycles of 95°C for 15 seconds and 60°C for 1 minute to allow for denaturation, annealing, and extension; and concluded with generation of a melt curve (CFX Connect 96 thermal cycler, Bio-Rad). Samples were run in duplicate for three independent experiments, with GAPDH as a reference gene to normalize differing levels of expression.