qPCR
U2OS cells were seeded on 10 cm plates under standard culture conditions
and incubated for 24-48 hours to reach 100% confluency. Once cells
reached confluency, RNA from cells were isolated using RNeasy Plus Mini
Kit (74134, Qiagen). Using the RNA, cDNA was synthesized using qScript
cDNA SuperMix and protocol from Quantabio (101414-102). The qPCR primer
cocktails were made using PerfeCTa SYBR Green FastMix (101414-276),
forward primer, reverse primer (Real Time Primers) and RNase/DNase free
H2O. In a 96 well qPCR plate, 18 µl of primer cocktail
was added and 2 µl of cDNA, accordingly. qPCR was run using BioRad cfx96
and fold gene expression was calculated.