B7-H4实时荧光定量PCR检测方法的建立及其在人胃癌组织中的初步应用

目的建立实时荧光定量PCR检测B7-H4的方法并初步应用于人胃癌组织。方法将B7-H4和内参GAPDH的PCR扩增产物经测序鉴定正确后克隆入载体pMD19-T,构建重组质粒标准品,并纯化、定量及梯度稀释,分别建立B7-H4和GAPDH的标准曲线,应用实时荧光定量PCR检测8例人胃癌组织中的B7-H4相对于GAPDH的表达情况。结果 B7-H4的最低检测拷贝数为5.27拷贝,线性范围5.27×101~5.27×107拷贝,标准曲线方程y=–3.1395x+41.805,直线回归相关系数r=0.994904,批间变异系数范围2.39%~3.59%,扩增效率108.2%;GAPDH的最低检测拷贝数为38.6拷贝,线性范围3.86×102~3.86×107拷贝,标准曲线方程y=–3.2436x+41.083,直线回归相关系数r=0.998913,批间变异系数范围2.26%~3.86%,扩增效率103.4%。8例人胃癌组织的B7-H4相对于GAPDHmRNA表达水平在0.044~0.888之间。结论荧光定量PCR检测B7-H4的方法具备较高的敏感性和特异性,且系统有良好的重复性和线性范围。

Quantification of B7-H4 with real-time fluorescent PCR and its preliminary application in gastric carcinoma

Objective To establish a real-time fluorescent polymerase chain reaction for quantifying B7-H4 and detect the expression of B7-H4 in human gastric carcinoma. Methods The B7-H4 and internal reference gene GAPDH fragments in correct sequence from RT-PCR were cloned into pMD19-T vector, and recombinant plasmids were purified and quantified by spectrophotometry. Standard curves was established by using a serial dilution of quantified plasmids in order to measure B7-H4 and GAPDH. Real-time fluorescent PCR was used to quantify the expression level of B7-H4 relative to GAPDH in 8 gastric carcinoma tissues.Results For B7-H4, the detection of the minimum copy number was 5.27 copies. The linear range of 5.27 × 10^1 - 5.27 × 10^7 copies of the standard curve equation （y = -3.1395 x ＋ 41.805） was found and the correlation coefficient for 0.994 904 was obtained. The inter-assay CV ranged from 2.39% to 3.59% and the amplification efficiency was 108.2%. For GAPDH, the detection of the minimum copy number was 38.6 copies. The linear range of 3.86 x 102 to 3.86 × 10^7 copies of the standard curve equation （y = -3.2436 x ＋ 41.083） was found and the correlation coefficient of 0.998 913 was obtained. The inter-assay CV ranged from 2.26% to 3.86% and the amplification efficiency was 103.4%. mRNA levels of B7-H4 relative to GAPDH in 8 gastric carcinoma tissues were between 0.044 and 0.888. Conclusion The real-time fluorescent quantitative PCR system established for quantifying B7-H4 is rapid, specific, sensitive and accurate. The standard system is repeatable and has a good linear range.