Real time fluorescent quantitative RT-PCR in determination of Dicer mRNA in primary hepatocellular carcinoma
- VernacularTitle:实时荧光定量RT-PCR检测原发性肝细胞癌中Dicer mRNA的表达
- Author:
Yulan WANG
- Publication Type:Journal Article
- Keywords:
Dicer;
carcinoma,hepatocellular;
real time fluorescent quantitative RT-PCR
- From:
Academic Journal of Second Military Medical University
2000;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a real time fluorescent quantitative reverse transcriptase polymerase chain reaction(RT-PCR) system for determining the expression of Dicer mRNA in human hepatoma cell lines and 20 samples of primary hepatocellular carcinoma(PHC)tissues.Methods: The specific primers,designed according to the complete sequence of Dicer mRNA,and the fluorescence dye SYBR Green Ⅰwere used for RT-PCR amplification.The fluorescence was monitored in a real time manner.The expression levels of Dicer mRNA in samples were calculated according to the standard curve and the nonspecific amplifications were excluded by melting curve analysis.The mRNA levels of Dicer were presented as the ratios of Dicer mRNA to 18S rRNA.Results: The linear detection range of this system was from 5?10~(2)to 5?10~(9)copies/?l and the coefficient of variation values for intra-experimental and inter-experimental reproducibility ranged from 4.13% to 19.72% and from 6.25% to(18.76%,) respectively.The expression levels of Dicer mRNA in HBV positive hepatoma cell line HepG2.2.15 or in HBV negative hepatoma cell line HepG2 were significantly lower(P
