Quantitative RT-PCR for Gene Expression

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed as described previously [19 (link)]. At endpoint, cells were rinsed 2× with PBS then TRIzol® (1 ml/well) was added and lysates were transferred to screw cap tubes. Chloroform (200 μl) was added and tubes were shaken to mix. Samples were centrifuged, the aqueous layer was transferred to a new tube and an equal volume of 70% ethanol (EtOH) was added. RNA was then extracted using RNeasy kits (Qiagen Inc. Venlo, Limburg, Nethlands). cDNA was made from 3μg DNase-1 (Promega, Madison, WI, USA) treated RNA using the Superscript III first strand synthesis kit (Invitrogen, Calrsbad, CA, USA). qRT-PCR was performed using Platinum SYBR Green qPCR SuperMix-UDG with Rox kit (Invitrogen). Standard curves were performed for all primers used; sequences and efficiencies are included in Additional file 2: Table S2. The level of gene expression was normalized to GAPDH at time zero or untreated conditions using the ΔC_t method [25 (link)].