建立SYBRGreen实时RT-PCR定量检测大鼠I型胶原mRNA水平方法

SYBR Green I-based real-time quantitative RT-PCR method for detecting mRNA level of type I collagen in rats

目的：基于双链嵌合荧光染料SYBR Green Ⅰ，建立一种快速、灵敏的检测大鼠Ⅰ型胶原mRNA表达水平的实时逆转录聚合酶链反应（RT-PCR）方法．方法：提取大鼠肝脏总RNA，RT-PCR扩增Ⅰ型胶原基因部分片段，将其克隆入pMD18-T载体用作参比品．基于SYBR GreenⅠ双链嵌合染料建立检测大鼠Ⅰ型胶原mRNA表达水平方法．其中对一系列连续稀释的参比品进行实时PCR分析，评价所建立方法的检测灵敏度；对PCR产物的熔解曲线进行分析评价其特异性．结果：重组质粒构建成功，经测序鉴定，目的片段已插入pMD18-T载体内．所建立的实时RT-PCR方法的最低检测限度为1个拷贝／反应，在每反应10^0～10^7拷贝范围内，CT值（C代表cycle，T代表threshold；CT值指荧光信号达到设定的阈值所经历的循环数）与起始模板浓度具有良好的线性关系，相关系数为0．991．结论：所建立的基于SYBR GreenⅠ双链嵌合染料的大鼠Ⅰ型胶原PCR检测方法具有敏感性高、特异性强和线性检测范围广等特点，适用于进一步的各种组织的大量样本检测．

AIM： To establish SYBR Green Ⅰ-based real-time quantitative RT-PCR method for detecting type Ⅰcollagen mRNA in rats. METHODS： Type Ⅰcollagen cDNA was amplified by RT-PCR method and cloned into pMD18-T vector, The recombinant plasmid was selected and identified by sequence analysis. It was then diluted pro rata as standard template, The mRNA expression of type Ⅰcollagen in rats was detected using real-time quantitative RT-PCR method with fluorescent DNA dye SYBR Green Ⅰ. RESULTS： The recombinant plasmid was established and confirmed as expected. The kinetics graph after real-time PCR detected even one molecule, The standard curve indicated the liner relationship between CT （cycle threshold） and template concentration. The correlation coefficient of external standard between the input copies and fluorescence intensity was 0. 991, CONCLUSION ： The established method for detecting type Ⅰcollagen mRNA in rats is sensitive and specific, Real-time quantitative RT-PCR based on SYBR Green Ⅰ is an excellent candidate as a standard detection method for large-scale application.