| Objective: A real-time fluorescence quantitative PCR method based on S-Poly(T)plus was developed to provide a more sensitive and convenient method for detecting serum mi R-122 and mi R-199.And to explore its application value in the diagnosis of hepatocellular carcinoma.Methods: 1.design S-poly(T)plus method to amplify specific primers and probes of mi RNA.2.S-poly(T)plus fluorescence quantitative PCR method was established to detect the tissue and serum mi RNA,and the experimental conditions were optimized,the linear range was established,and the sensitivity and repeatability were evaluated.3.From February 2017 to December 2017,50 patients with primary hepatocellular carcinoma in the First Affiliated Hospital of Fujian Medical University were selected as the experimental group.At the same time,the sex and age were matched with the experimental group,and the healthy people with normal physical examination and no family history of tumor were selected as the control group.The expression levels of mi R-122 and mi R-199 in serum of each group were detected by S-poly(T)plus fluorescence quantitative PCR,and compared with Poly(A)plus tail method,and ROC curve analysis was performed.4.According to the level of AFP in the experimental group,the levels of AFP were divided into group A(AFP<20),group B(20<APF<400),group C(AFP>400),and the correlation between the level of serum mi R-122 and mi R-199 expression and AFP level was analyzed.Result: 1.A method for detecting micro RNA based on S-poly(T)plus fluorescence quantitative PCR was established.Micro RNA was detected by S-poly(T)plus and Poly(A)plus tail method.The Ct values of each group were as follows: experimental group mi R122(26.73±2.48)and(32.84±2.28)(P<0.05),mi R122(27.87±2.66)and(32.12±1.84)(P<0.05)in the control group,and the experimental group(33.62±1.6)and(34.53±1.13),respectively.In the group of mi R199(30.71±3.21)and(35.25±0.83)(P<0.05),the experimental group was cel-mi R-54(29.43±1.37)and(33.79±0.77)(P<0.05),the control group was cel-mi R-54(31.88±2.39)and(33.38±0.61)(P<0.05).The comparison results of the above corresponding groups showed that the sensitivity of S-poly(T)plus method was higher than Poly(A)plus tail.Law.2.The CV value of 2.S-poly(T)plus method was 2.59%,less than 3.52% of Poly(A)plus tail method.The linear correlation coefficient R2(0.984)of S-poly(T)plus method is larger than that of Poly(A)plus tail method R2(0.957).3.we used S-poly(T)plus to detect mi R-122 and mi R-199 in the experimental group and the control group.The experimental group mi R-122(10.89±11.99),the control group mi R-122(2.11±3.26),the level of serum mi R-122 in patients with hepatocellular carcinoma was significantly higher than that in healthy volunteers,P<0.01.The experimental group mi R-199(0.2±0.2),the control group mi R-199(0.92±0.93),the serum level of mi R-199 in patients with hepatocellular carcinoma was significantly lower than that in healthy volunteers,P<0.01.4.The ROC curve analysis shows that the area under the curve(AUC)of mi R-122 is 0.82(95%CI 0.732-0.908,P<0.001,cut-off is 1.32,the sensitivity is 81.6%,the specificity is 68.2%),and the area under the curve of mi R-199 is 0.807(95%CI 0.714-0.900,P<0.001,0.226,73%,and 71.4%).5.The expression level of serum mi R-122 increased gradually with the increase of AFP in A,B and C subgroups(P<0.05).The expression level of serum mi R-199 decreased gradually with the increase of AFP in A,B and C subgroups(P<0.05).Conclusion: 1.A method for the detection of micro RNA based on S-poly(T)plus fluorescence quantitative PCR is established.This method has higher sensitivity,repeatability and better linear correlation coefficient than Poly(A)plus tail method.2.Serum mi R-122 and mi R-199 are expected to be an auxiliary diagnostic index for hepatocellular carcinoma. |
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