Clinical Implications and Prognostic Values of Prostate Cancer Susceptibility Candidate Methylation in Primary Nonmuscle Invasive Bladder Cancer

Table 2

PRAC primers used for pyrosequencing analysis.

Genes	PRAC
Forward (5′-3′)	GGATTTTGGTTTTTATTTTTGTAGA
Reverse (5′-3′)	(biotin)-CTCACCCTTCCCTTATTTC
Sequencing primer (5′-3′)	TGTTTTTTTTTTTAATAAGGTAAAT
Amplicon location relative to TSS	−35–209
Sequence to analyze	TGATCGGTGGGCCAAGGGCGTTATCGACGGATCG
Product size	244 bp

Primer was designed using NCBI Reference Sequences build version 36.1.
The PCR reaction contained 0.01 μM each of primers and Bioneer Taq (Bioneer, Daejeon, Korea), and 20 ng of bisulfite-treated DNA. The thermocycling parameters were as follows: denaturation at 94°C for 5 minutes, followed by 45 cycles at 94°C for 30 seconds, annealing at 55°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes.
bp: base pairs; PRAC: prostate cancer susceptibility candidate; TSS: transcription start site.