[Detection of methylation in hepatocellular carcinoma using SYBR Green fluorescent quantitative PCR].
OBJECTIVE
To establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application.


METHODS
Following bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen.


RESULTS
The amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation.


CONCLUSIONS
SYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.
The Key Laboratory of Artificial Cell, the Third Central Hospital of Tianjin, Tianjin 300170, P.R. China.
Bin
B
Yang
Cheng
C
Lou
Yingtang
Y
Gao
Zhi
Z
Du
Wenqin
W
Song
D000199:Actins / Q000235:genetics; D001706:Biopsy; D002138:Calibration; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation; D005240:Feasibility Studies; D005260:Female; D005453:Fluorescence; D019942:Genes, p16; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D008875:Middle Aged; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D000199:Actins