实时荧光定量PCR检测肝癌组织中早期B细胞因子3 mRNA表达水平

Detection of EBF3 mRNA in Primary Hepatocellular Carcinoma by Real-time Fluorescence Quantitative Method

目的建立SYBR Green I实时荧光定量RT—PCR（FQ—RT—PCR）检测肝组织中早期B细胞因子3（early B—cell factor 3，EBF3）mRNA含量的方法，探讨EBF3 mRNA在肝癌中的检测意义。方法在EBF3基因第8和第9外显子区，在内参β2M第1和第2外显子区设计特异性引物，实时检测PCR产物的荧光强度，以各自标准品建立标准曲线，由软件自动计算出待测样本中EBF3及β2M mRNA含量，以EBF3 mRNA和β2M mRNA含量的比值进行EBF3 mRNA表达水平评价。结果FQ—RT—PCR检测EBF3 mRNA含量线性范围为3．08×10^7～3．08×10^12 copies／L，批内和批阅变异系数分别为8．6％和13．7％。18例肝癌和配对远端肝组织中EBF3 mRNA与β2M mRNA的对数比值分别为0．52±0．17和0．28±0．23，差异有统计学意义0=3．56，P=0．0011）。结论FQ—RT—PCR检测EBF3 mRNA含量的方法具有较好的检测灵敏度和重复性，适用于临床研究。

Objective To establish a SYBR Green I real-time fluorescence quantitative polymerase chain reaction（FQ-RT- PCR） for detecting early B-cell factor 3（EBF3） mRNA in primary hepatocellular carcinoma （HCC）,and investigate the significance of EBF3 mRNA level in HCC. Methods The upper and lower specific primers of EBF3 and β2 microgluobulin （β2M） were designed by the eighth and ninth extron sequence of the EBF3 or by the first and second extron sequence of the β2M respectively. The fluorescence of the PCR product was detected continuously during amplification. According to the standard curve created by plasmid DNA,the expression levels of EBF3 mRNA in liver cancerous tissues and distant liver noncancerous tissues were determined by software and the results were presented as the ratios of EBF3 MRNA to β2M mRNA. Results The detection range of the assay was from 3.08 × 10^7 copies/L to 3.08× 10^12 copies/L. The coefficients of variation values of both intra-experimental and inter-experimental reproducibility were 8.6% and 13.7%. The ratios of EBF3 mRNA to β2M mRNA in 18 liver cancerous tissues （0. 52±0.17） were significantly higher than those in distant liver noncancerous tissues （0. 28±0. 23 ,t= 3.56 ,P= 0. 0011 ）. Conclusion This assay has high sensitivity and reproducibility. It is suitable for measurement of the expression level of EBF3 mRNA in clinical research.