实时荧光定量RT-PCR在小鼠及人G蛋白mRNA检测中的应用

用自行设计的TaqMan双标探针和扩增引物建立检测鸟苷酸结合蛋白(G-protein)mRNA的实时荧光定量RT-PCR技术.用G蛋白纯品绘制定量标准曲线,实时荧光定量PCR仪检测ECV304细胞和小鼠G蛋白mRNA水平.10μmol/LGqαmRNA反义寡核苷酸(GqαODN)作用ECV304细胞24h后,Gqα的mRNA表达显著下降(3.18×108±1.75×108拷贝下降到1.44×106±4.82×105拷贝),48h和72h下降更明显;Gsα和Gi2α的mRNA表达变化不显著.10μmol/LGsαmRNA反义寡核苷酸(GsαODN)作用ECV304细胞24h后,Gsα的mRNA表达显著下降(2.97×108±2.68×107拷贝下降到4.16×106±2.00×106拷贝),48h和72h下降更明显;Gqα、Gi2α表达变化不显著.小白鼠油酸致伤后6h,GqαmRNA表达显著下降(1.16×108±8.73×106拷贝下降到3.30×106±1.68×106拷贝),24h下降更显著(9.32×107±1.47×107拷贝下降到4.14×106±1.67×106拷贝);Gsα和Gi2α表达变化的趋势同GqαmRNA.结果准确可靠,重现性好.说明建立的实时荧光定量RT-PCR方法成功地实现了对ECV304细胞和小白鼠肺组织G蛋白不同亚型不同丰度基因表达的检测.

Application of Real Time Fluorescence Quantitative RT-PCR in Detection of Mouse and Human G-Protein mRNA

Guanine nucleotide binding proteins(G-protein) are glycoproteins anchroed on the cytoplasmic cell membrane.The application of real-time fluorescence quantitative RT-PCR was presented for detecting six mRNA of G-protein subunits from mouse and human.Based on GenBank,six double-labeled TaqMan primers and corresponding probes were designed,following construction of standard curve for HGq cDNA.Real time fluorescence quantitative RT-PCR was adopted to detect the mRNA levels of G-protein in ECV304 cells and mouse lung tissue. The Gqα mRNA expression level in ECV304 cells dramatically decreased at 24 hours after treated by 10 μmol/L Gqα ODN, from 3.18×10 8±1.75×10 8 copies to 1.44×10 6±4.82×10 5 copies, it was more apparent at 48 hours and 72 hours after treatment. The mRNA expression levels of Gsα and Gi2α showed no obvious change. With the same treatment,the Gsα mRNA level also dramatically decreased at 24 hours after treated by 10 μmol/L Gsα ODN, from 2.97×10 8±2.68×10 8 copies to 4.16×10 6±2.00×10 5 copies, and decreased more sharply after 48 hours and 72 hours. Six hours after the mouse was injured by oleic acid, the expression level of Gqα mRNA in lung tissue dramatically decreased from 1.16×10 8±8.73×10 6 copies to 3.30×10 6±1.68×10 6 copies, which became more obviously 24 hours after treatment, from 9.32×10 7±1.47×10 7 copies to 4.14×10 6±1.67×10 6 copies, their trend of Gsα and Gi2α mRNA levels were same as Gqα mRNA .These results were determined reproducibly, which suggested that the real-time RT-PCR method offered a valid detection for gene expression of different subtypes and different abundances in the mouse lung and cultured cell lines.