Quantitative Reverse-Transcription Polymerase Chain Reaction

CaP and BP tissues were Dounce homogenized, tissue lysates were passed through QIAshredder columns, and RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Genomic DNA contamination was assessed using qualitative PCR using intron-spanning glyceraldehyde 3-phopsphate dehydrogenase (GAPDH) primers. Genomic DNA contamination was removed using the DNA-free DNA removal kit (Life Technologies, Inc, Carlsbad, CA). RNA was analyzed using qualitative PCR after DNAase treatment to confirm genomic DNA was removed.

First strand complementary DNA (cDNA) was generated using 2 mcg of RNA with the High-Capacity cDNA Reverse Transcription Kit (10× RT Buffer, 10× Random Primers, 25× 100 mM deoxyNTP mix, and 50 U/mcL MultiScribe-Reverse Transcriptase; Applied Biosystems, Foster City, CA) with RNAse inhibitor in a reaction volume of 10 mcL as directed. qRT-PCR primers were designed using the IDT PrimerQuest design tool (Integrated DNA Technologies, Coralville, IA; primer sequences are listed in supplemental eTable 4).

qRT-PCR reactions were mixed using 12.5 mcL SYBR Green PCR Master Mix (Applied Biosystems); 1 mcL (10 mM forward and reverse primers) and 2.5 mcL (100 ng/mcL) cDNA (250 ng final concentration); and 10 mcL ddH₂O, for a final reaction volume of 25 mcL. Reactions were performed in 96- well plates. Gene expression was analyzed using the 7300 Real Time System (Applied Biosystems). The qRT-PCR reaction profile was 95°C for 30 seconds, 60°C for 30 seconds repeated 39 times, 95°C for 5 seconds, and melt curve of 65°C to 95°C. All procedures were conducted in Dr. Mohler’s laboratory, and the technique was performed with 3 technical replicates and 1 biological replicate, because BP and CaP and tissues were collected from 1 patient. Quatification cycle (Cq) values were normalized against β₂ microglobulin (B2M). B2M was selected because BP and CaP or B2M gene expression levels remain unchanged during androgen deprivation therapy. Cqs for no reverse transcriptase control and no template controls were reported as undefined. Relative gene abundance was calculated using 2^–(Cq–B2M).