内参基因β-actin质粒标准品的构建

Plasmid construction of reference gene beta-actin

为构建检测内参基因β-actin mRNA表达水平差异的标准品,以成人外周血细胞的总RNA为模板、Random 6 mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人β-actin基因相应的cDNA片段,构建PMD-18-β-actin重组质粒,鉴定测序后,用荧光定量PCR制作标准曲线。结果外周血提取的总RNA完整性良好,构建的β-actin质粒经PCR扩增后得到一186bp的清晰条带,测序结果与目的片段完全一致,且质粒的原始浓度为2.39×1013 copies/mL,倍比稀释至2.0×104copies/mL均能得到良好的标准曲线(R2=1),提示构建β-actin基因荧光定量PCR标准质粒成功。

To clone human β-actin cDNA and using it as the standard for real-time quantifying β -actin mRNA. Total RNA was extracted from the adult peripheral blood cells and reverse transcribed to cDNA using Random 6 mers as primer. Amplified β -actin cDNA was ligated with pMD 18-T simple vector and transformed to bacteria JM109. Plasmids were extracted from positive clones and identified with polymerase chain reaction （PCR）, and then were subjected to sequencing. Total RNA extracte4 from the adult peripheral blood cells was perfected. The PCR of β -actin plasmid was a 186 bp clear stripe, and the DNA sequencing of β -actin was according with aimed fragment （100%）. The original concentration was 2.39 × 10^13 copies/mL, and then diluted to 2.0 × 10^4 copies/mL with multiple proportions, perfected standard curve could be all received （R2= 1）. The recombined plasmids containing human β -actin cDNA have been constructed successfully.