SYBR Green实时荧光定量PCR检测水牛体细胞组蛋白乙酰化相关基因mRNA表达

Detection of histone acetylation related gene expression in buffalo somatic cells by SYBR GreenⅠreal-time fluorescence quantitative PCR

以检测水牛成纤维细胞中组蛋白乙酰转移酶(HAT1)和脱乙酰化酶(HDAC1)转录活性为例,对SYBRGreen实时荧光定量PCR技术平台的建立进行了探讨。结果显示,适宜的引物,高质量RNA反转录得到的cDNA以及合适的PCR体系为SYBR Green实时荧光定量PCR能否成功的关键因素。本试验中3对引物的灵敏度高,其反应性能完全满足SYBR Green实时荧光定量PCR的要求。2个目的基因HAT1、HDAC1的扩增效率与参照基因H2A接近,试验结果可用2-△△Ct计算法进行分析。当cDNA模板量为0.5μL(相当于总RNA 50 ng),引物为1μmol/L时,SYBR Green实时荧光定量PCR能高效获得特异性强的目的产物。

Histone acetylation modification participates in the regulation of gene expression by altering structure of chromatin. The real-time fluorescence quantitative PCR is one of the most effective,accurate and sensitive technology for investigating gene expression level. Here we describe the details of detecting buffalo HAT1 and HDAC1 ex pression as the example for constructing a platform of real-time fluorescence quantitative PCR using, SYBR Green Ⅱ as dye. The transcript effective, primer design, the concentrations of primer and template , and 2^△△△, quantitative method were investigated. The homology sequence of bovine guide the proper primer design and the result was confirmed by the PCR product sequencing. The primers are sensitive enough and fulfilled for SYBR Green I real time fluorescence quantitative PCR. It is found that effective transcript template 0.5 μL（equal to 50 ng total RNA） and 1 μmol/l. high effective primer is able to give the best SYBR Green real time fluorescence quantitative PCR results.