Quantitative PCR was used to assess SelP mRNA expression. 293-EcR and EcR-GR cells were treated with 10 μM ponasterone A 24 hours prior to mRNA collection, and 10 nM dexamethasone was then added at 8 or 16 hours prior to mRNA purification. Vehicle treatments with ethanol (EtOH) or dimethyl sulfoxide (DMSO) were used as controls. The Qiagen RNeasy Mini Kit was used to collect and purify mRNA from cells. First strand cDNA was synthesized using Superscript III reverse transcriptase and these cDNA samples were run in triplicate as 1:5 dilutions. Standards were run in duplicate at concentrations between 103 to 108 copies/μl and β2 microglobulin was run as a reference gene. The SEPP1 amplicon consisted of the 100 bp spanning the final intron of the genomic sequence. The primer pair 5′-TTCGGGCAGAGGAGAACA-3′ and 5′-CTGGCACTGGCTTCTGTG-3′ were used to amplify this region. Average threshold copy number was used to calculate changes in expression level as compared to vehicle treated controls.
