Establishment of real-time fluorescence quantitative polymerase chain reaction analysis assay quantification for hepatocyte growth flactor mRNA expression and its clinical relevance in lymphoma / 中华检验医学杂志

ABSTRACT

Objective To construct quantitative standard for quantification of hepatocyte growth factor (HGF) mRNA and establish its real-time fluorescence quantitative(FQ)-PCR assay to estimate its clinical relevance in lymphoma.Methods Recombinant plasmid Was constructed with target cDNA obtained from isolated total RNA by RT-PCR After PCR products were identified and purified,recombined plasmids were quantitated and then acted as quantitative standard.A new real time FQ-PCR analysis system Was established with the second pair of primers and the probe after amplification condition and the concentrations of components were optimized.HGF mRNA expressions in 47 lymohoma cases[11 Hodgkin disease(HD) cases,36 non-Hodgkin lyphoma(NHL)cases.among these patients,36 patients in remission while 11 patients without remission ] were analyzed quantitatively,and its specificity and sensitivity for lymphoma diagnosis were evaluated by receiptor operation character(ROC)curve method.Results HGF mRNA quantitative standard was constructed successfully.and its real time FO.PCR analysis system Was established combined with hot.start PCR and down.touch PCR technique. According to slope of standard curve (-3.513)and correlation cofficient(0.999),amplification efficiency of the system was 92.6%.Coefficient variation of intra-assay,intra-day and inter-day-assay were 2.1%,4.0% and 6.8%,respectively.Sensitivity of FQ-PCR Was 2 eopies/μl.Expressions of HGF mRNA in lymphoma group Was higher than that in control group(6.425±2.172 and 0.317±0.192,respectively,t=15.883,P<0.001),and its expressions in remission group was lower than no remission group(6.157±1.712 and 7.59l ±1.184,respectively,t=2.768,P<0.05).However,there Was not difference of HGF mRNA level between group HD and group NHL(P>0.05).According to ROC analysis,its sensitivity and specificity were 93.6% and 100% when cutoff value for lymphoma clinical diagnosis Was 3.136.Conclusion HGF mRNA'8 quantitative standard and its real time F9-PCR analysis system have been successfully constructed,and it can be used for quantitative detection of its mRNA expression in lymphoma.