Real time fluorescent quantitative RT-PCR in determination of Dicer mRNA in primary hepatocellular carcinoma / 第二军医大学学报

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 I were 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 × 102 to 5 × 109 copies/μl 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<0.05) compared to those of the normal liver cell line WRL-68; and those in PHC tissues were lower compared to those in the corresponding adjacent tissues of PHC (P<0.05). Conclusion: Real time fluorescent quantitative RT-PCR has good sensitivity, specificity and reproducibility in determining Dicer mRNA levels. Determination of Dicer mRNA levels in hepatoma cell lines and PHC tissues lays a good theoretical foundation for further studying the mechanism of PHC.