Superscript 3 first strand system kit

Total RNA was isolated from G361 and MeWo cells by using the RNeasy Mini kit (Qiagen Inc.) and converted to first-strand cDNA using the SuperScript III First-Strand System Kit (Thermo Scientific). Real-time RT-qPCR was performed with SYBR Green Real-time PCR Master Mixes (Thermo Scientific) according to the manufacturer’s instructions. The primers used for RT-qPCR are listed in Additional file 14: Table S1. All mRNA values were normalized to GAPDH mRNA levels, and all reactions were run in triplicate.

Total RNA from HDFs were isolated using the SV Total RNA Isolation System following the manufacturer’s instructions (Promega, Madison, WI, USA), and cDNA synthesis was performed using the SUPERSCRIPT III First-Strand System kit (Thermo Fisher Scientific, Waltham, MA, USA). Hot start PCR was performed using 2 ng cDNA in a total volume of 50 μL containing DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA).
The primer sequences were as follows: human-Panx1, Forward: (5′-GCTATTACTTCAGCCTCTCC-3′), Reverse: (5′-CAGTATCTCCACCAAGAACC-3′); human-Panx3, Forward: (5′-AGGGCTGCTAAGTGATGAGA-3′), Reverse: (5′-GAGGTGTTTGGGTTTTGAGG-3′). PCR reactions were performed using 40 cycles and amplified PCR products were visualized in a 3% agarose gel.

LNCaP, DU145, and PC3 cells were treated with 10 μM 5-Aza-CdR for 1, 2, 3 and 4 days, and the medium containing 5-Aza-CdR was changed daily. Total RNA was isolated from untreated or 5-Aza-CdR-treated cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. cDNA was synthesized from 2 μg of total RNA using first-strand cDNA using the SuperScript III First-Strand System Kit (Thermo Fisher Scientific, Waltham, MA, USA). The real-time RT-qPCR was performed with SYBR Green Real-time PCR Master Mixes (Thermo Fisher Scientific, Waltham, MA, USA) using Agilent Aria 1.71 software according to the manufacturer’s protocol. The primers used for RT-qPCR are listed in Supplementary Table S1. All RT-qPCR reactions were run in triplicate, and results were normalized to β-actin mRNA levels.