Plus real time pcr system

Total RNAs of all samples were extracted using TRIzol reagent (Invitrogen, United States), and genomic DNA contamination was removed by DNase I (Promega, United States). The RNA yield was determined using NanoDrop ND-2000 (Thermo Fisher Scientific, United States), and integrity was checked on a 1% agarose gel. The cDNA was synthesized using oligo-dT primers and Superscript RT-III (Takara, JP). Quantitative real-time PCR (qRT-PCR) was performed using a Plus Real-Time PCR System (Applied Biosystems, United States). SYBR Prime ScriptTM RTPCR kit (Takara, JP) was used for mRNA qRT-PCR. Data were analyzed using the relative 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link); Schmittgen and Livak, 2008 (link)). The primers for qRT-PCR are listed in Supplementary Table 3.

Protein–protein interaction data for prostate cancer-related genes were obtained from the STRING database. A network was visualized using the software Cytoscape (3.9.1 version; https://cytoscape.org/index.html)^{123 (link)}, and the core interacting protein network was extracted.
Total RNA from all samples was extracted using the TRIzol reagent (Invitrogen, USA), and genomic DNA contamination was removed using DNase I (Promega, USA). RNA yield was measured using a NanoDrop ND-2000 (Thermo Fisher Scientific, USA), and integrity was assessed on a 1% agarose gel. cDNA was synthesized using oligo-dT primers and SuperScript III (Takara, JP). Real-time Quantitative PCR (RT-qPCR) was performed using the Plus real-time PCR system (Applied Biosystems, USA). mRNA RT-qPCR was carried out using the SYBR Prime ScriptTM RT-PCR kit (Takara, JP). Data were analyzed using the relative 2^−ΔΔCT method^{124 (link)}. RT-qPCR primer sequences are listed in Supplementary Table S22.