血清miRNA-221-3p标准品的制备及其绝对含量测定  

Preparation of serum miR-221-3p standard substance and determination of its absolute content

作　　者：邱玲 冯建国 杨继梅 李茂 王晓斌 

机构地区：[1]西南医科大学附属医院麻醉科,四川泸州646000 [2]西南医科大学麻醉学研究室,四川泸州646000

出　　处：《西南医科大学学报》2017年第6期524-528,共5页Journal of Southwest Medical University

基　　金：国家自然科学基金(81271478);四川省科技厅-泸州市-泸州医学院联合资助项目(14JC0093)

摘　　要：目的:构建miRNA-221-3p的标准品,并检测其血清绝对含量,为今后进一步明确检测血清miRNA-221-3p在辅助判断疾病中的潜在价值和意义。方法:设计miRNA-221-3p特异性引物,提取样品血清总RNA,反转录获得含miRNA-221-3p特异性引物c DNA。通过PCR获得含有miRNA-221-3p的DNA片段,然后置入到pc DNA3.1质粒中。经测序确定重组质粒pc DNA3.1(pc DNA3.1-miRNA-221-3p)构建正确。通过PCR从pc DNA3.1-miRNA-221-3p中获得含有T7启动子和miRNA-221-3p的基因片段,然后用体外转录得到miRNA-221-3p的标准品,最后计算出标准品的浓度(copies/μg RNA)。取miRNA-221-3p标准品反转录进行梯度稀释实时荧光定量PCR,根据标准品的浓度和CT值绘制标准曲线。结果:miRNA-221-3p重组质粒构建成功,体外转录得到纯化的miRNA-221-3p标准品。标准曲线公式为:Y=-0.255X+12.543,相关系数R2=0.9999,X代表Ct值,Y代表样品的含量(copies)的lg值。结论:成功获得血清miRNA-221-3p标准品,miRNA-221-3p标准品绝对含量(copies/μg RNA)等于3.49214E+12e-0.5871CT值。Objective To prepare serum miR-221-3p standard substance, to determine its absolute content, and to investigate the value and significance of serum miR-221-3p determination in disease diagnosis. Methods： The specific primer of miR-221-3p was designed, total serum RNA was extracted, and then reverse transcription was performed to obtain cDNA that contained the specific primer of miR-221-3p. PCR was performed to obtain DNA fragments containing miR-221-3p , which were inserted into pcDNA3.1 plasmid. Sequencing was used to confirm whether the recombinant plasmid pcDNA3.1-miR-221-3p was correctly constructed. Then PCR was used to obtain gene fragments containing T7 promoter and miR-221-3p from pcDNA3.1-miR-221-3p , and in vitro transcription was performed to obtain miR-221-3p standard substance. The concentration of miR-221-3p standard substance （copies/^g RNA） was calculated. Gradient dilution and quantitative real-time PCR were performed for miR-221-3p standard substance, and a standard curve was plotted based on concentration and CT of standard substance. Results： The recombinant plasmid of miR-221-3p was constructed successfully, and the purified miR-221-3p standard substance was obtained by in vitro transcription. The standard curve formula was Y = - 0.255X ＋ 12.543 （R2 = 0.9999）, with X standing for threshold cycle and Y for the lg value of content. Conclusions： Serum miR-221-3p standard substance is obtained successfully, and the absolute content of miR-221-3p standard substance equals to 3.49214E ＋ 12e-0.5871 Ct.

关 键 词：血清 miRNA-221-3p 标准品 绝对含量 

分 类 号：R749[医药卫生—神经病学与精神病学]