Comprehensive qPCR Workflow for Gene Expression Analysis

RNA was extracted using QIAshredder (Qiagen) and RNeasy Kit (Qiagen) with on-column RNase-free DNase treatment (Qiagen) according to manufacturer’s protocol. 2 µg of RNA was reverse transcribed using SuperScript IV (Thermo Fisher) and manufacturers protocol. qPCRs for AR, ERα, PGR, RUFY3, HPRT1, TFF1, PPL, CDKN1A, PCDH19, NOVA1, TET3, Gapdh, Tff1, Rara, Nrip1, Hsd11b2, Ppl, Pip, Cdkn1a, Pmepa1 and Pcdh19 were performed using TaqMan probes (Thermo Fisher, see Supplementary Table 6) with Taqman Gene Expression Master Mix (2×) (Thermo Fisher) and diluted cDNA. qPCR was performed using the following cycling conditions: hold at 50 °C for 2 min and 95 °C for 10 min, initial denaturation at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. qPCRs for GREB1, HSD11B2, PSA and HPRT1 were performed using previously published primers (see Supplementary Table 7) with Power SYBR Green Master Mix (Thermo Fisher) and diluted cDNA [17 (link), 50 (link)–52 (link)]. qPCR was performed using the following cycling conditions: hold at 95 °C for 10 min, 40 cycles of 95 °C for 15 s 60 °C for 1 min, melt curve: 95 °C for 15 s, 60 °C for 30 s and 95 °C for 15 s. Data was acquired using StepOne Real-Time PCR System and software V 2.0 (Applied Biosystems) and expression of the target gene was normalized against HPRT1 or Gapdh.