Quantitative PCR Analysis of AR Variants

The cDNAs were synthesized using the superscript III pre-amplification system (Invitrogen) after the total RNA was extracted from the cells using a TRIzol reagent (ambion, Life technologies, Carlsbad, CA, USA). The ARv7 primers (5′-GGAAATGTTATGAAGCAGGGATG-3′ and 5′-GGTCATTTGAGATGCTTGCA), ARFL primers (5′-TGCAGCCTATTGCGAGAGA-3′ and 5′-TGATCTCTGCCATCATTTCC), and 18S (5′-ACCGCAGCTAGGAATAATGGA-3′ and 5′-GCCTCAGTTCCGAAAACCA-3′) were used according to the previous report [50 (link)]. The qPCR reactions were performed in a 12 μL reaction volume that consisted of 6 μL of 2 x iQ^TM SYBR Green supermix (Bio-Rad Laboratories, Foster City, CA, USA), 1 μL of the cDNA sample, 1 μL of gene-specific primers (5 μM), and 4 μL of H₂O. For the MALT1 and NDRG1 assays, TaqMan^TM gene expression master mix and FAM dye-labeled TaqMan MGB probes for human MALT1 (Hs01120052_m1), NDRG1 (Hs00608387_m1), and β-actin (Hs01060665_g1) from Thermo Fisher Scientific Inc. (Vilnius, Lithuania) were used as described previously [35 (link)]. The qPCR analysis was performed using a CFX Connect Real-PCR system (Bio-Rad Laboratories, Foster City, CA, USA). The cycle threshold (Ct) values for the target genes were normalized against the 18S or β-actin control probe to calculate the mean cycle threshold (ΔCt) values using the Bio-Rad CFX manager 3.1 (Bio-Rad Laboratories, Foster City, CA, USA).