| ObjectiveTo develop fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) for quantification of iASPP mRNA, we used TaqMan probe and detected its level in hepatocellular carcinoma (HCC), adjacent liver tissue ,normal liver tissue and hepatoma cell lines (HepG2 and SMMC-7721 ).The results were linked with clinical indicators in this study to analyze the relationship between iASPP and HCC, and explore the possible functions of iASPP gene in HCC development and progression.Methods①According to iASPP mRNA sequence(NM006663) and the principle of TaqMan probe quantitative assay, the specific primers were designed using primer express 2.0.At the same time, one special TaqMan probe of iASPP was also designed, which can combine with the PCR product.②TaqMan probes complemented the templates were added to the reaction system. According to the standard curves created by a 10-fold dilution series of cDNA concentration and the values of their cycle (Cycle threshold, Ct), the expression level of target genes in liver cancer tissue has been determined. Evaluation to this real-time quantitative PCR method also has been carried on.③The total RNA isolated from human HCC, adjacent liver tissue, normal broken liver tissue and two hepatoma cell lines (HepG2 and SMMC-7721 ), was reversely transcribed into cDNA. Meanwhile housekeeping geneβ-actin was used as internal reference. The real-time fluorescence quantitative PCR method was used to analyze the expression level of iASPP gene. Results①FQ-RT-PCR for quantification of iASPP mRNA was successfully established. The efficiency of PCR is above 95%. The repetition is great.(The intra– and inter- assay coefficient variation was 2.2～3.1% and 2.0%).②iASPP mRNA was detectable in 82.6% of the tumors,47.8% of adjacent liver tissue, 16.7% of normal liver tissue and SMMC-7721 but was not detectable in HepG2;③The expression level of iASPP in hepatocellular carcinoma (HCC) was much higher than that of normal liver tissue and adjacent liver tissue group (P<0.05).④The different expression of iASPP mRNA in HCC was related to the tumor size and the pathological grade(P<0.05). The upper pathological grade or larger tumor, the expression level of iASPP is higher.Conclusions①FQ-RT-PCR for the measurement of iASPP mRNA is a sensitive,specific and rapid method,and it can be a useful technique for the quantification of iASPP mRNA.②iASPP ,high expressed in HCC, may inhibit the apoptotic function of p53, reduce the number of the apoptotic cells and neoplasia.③The expression of iASPP mRNA in HCC was related to the tumor size and the pathological grade, indicates iASPP expression maybe the reference guideline for prognosis and malignant degree in HCC.
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