The use of real-time quantitative polymerase chain reaction to detect hypermethylation of the CpG islands in the promoter region flanking the GSTP1 gene to diagnose prostate carcinoma.
OBJECTIVE
We developed a real-time, quantitative, methylation sensitive polymerase chain reaction (PCR) protocol to analyze hypermethylation of the CpG islands in the promoter region of the pi class glutathione-S-transferase gene GSTP1 in prostate cancer tissue.


METHODS
A total of 21 prostate cancer and 72 benign prostate hyperplasia (BPH) tissue samples were analyzed. Genomic DNA was digested with restriction enzyme, followed by real-time quantitative PCR amplification. Cycle threshold values were used to determine whether cancer genome was present in these tissues. A cutoff cycle threshold value of 35 was arbitrarily assigned. Samples with a cycle threshold of 35 or less were considered positive for prostate cancer. Conventional nested PCR was also performed for comparison.


RESULTS
The mean cycle threshold values plus or minus standard deviation in prostate cancer and BPH cases were 30.12 +/- 2.88 and 37.77 +/- 2.72, respectively. All prostate cancer samples analyzed showed positive results, while 5 of the 72 BPH samples tested positive. Conventional nested PCR data indicated that 19 of 21 prostate cancer cases were positive for the methylation change, while 71 of 72 BPH cases tested negative. The test limitations of real-time PCR and the nested PCR protocols were determined to be 0.048 and 0.64 ng. DNA, respectively.


CONCLUSIONS
We established a novel protocol for detecting the methylation change in the 5' regulatory sequence flanking the GSTP1 gene. The sensitivity of this protocol was superior to that of conventional nested PCR. The data also suggest that this novel protocol may accurately discriminate prostate carcinoma from BPH.
Chu
Da-Chang
DC
Department of Clinical Pathology and Division of Urology, Chang Gung Memorial Hospital and School of Medical Technology, Chang Gung University, Tao-Yuan, Taiwan, Republic of China.
Chuang
Cheng-Keng
CK
Fu
Jin-Bao
JB
Huang
Hsien-Siang
HS
Tseng
Ching-Ping
CP
Sun
Chien-Feng
CF
D003199:Computer Systems; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000175:diagnosis* / Q000235:genetics*
D007527:Isoenzymes; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase