巴马香猪CYP3A29 mRNA表达的TaqMan定量方法学建立

目的建立巴马香猪CYP3A29m RNA表达的TaqMan定量方法。方法通过RT—PCR、TA克隆等技术制备CYP3A29-pMDl8-T和β-actin—pMDl8-T实时荧光定量标准品，并以CYP3A29-pMDl8-T标准品为模板，对不同浓度Mg^2＋与探针组合的TaqMan反应体系进行筛选，TaqMan PCR方法，并对其有效性及重复性进行评价。结果筛选到的TaqMan PCR方法为：PCR总体积10μL，其中含l×Buffer、5．5mmol／L Mg^2＋、0．8mmol／L dNTPmix、0．3μmol／L上下游引物、0．2U/μL rTaq、0．1 μnoL／L探针、1μL模板，热循环条件为94℃ 5 min→40cycles（94℃15s→60℃45s→读板）。结论建立了有效测定巴马香猪CYP3A29 mRNA拷贝数浓度的TaqMan PCR方法，通过该方法分别定量CYP3A29和β-actln mRNA的拷贝数浓度，即可求出CYP3A29 mRNA的表达水平。

Establishing quantitative TaqMan method for determining expression level of CYP3A29 mRNA in Bama miniature pig's tissues

Objective：establishing quantitative TaqMan method for determining expression level of CYP3A29 mRNA in Bama miniature pig＇s tissues. Methods： CYP3A29-pMD18-T and β-actin-pMD18-T standards were prepared by RT-PCR and TA cloning technologies, and CYP3A29-pMD18-T standards were further used as template to screen out TaqMan PCR system that comprising different concentration of Mg^2＋ and probe. So TaqMan PCR method were established and it＇s validity and repeatability were evaluated, too. Results：The following TaqMan PCR method is available： 10 μL PCR system were comprised of l×Buffer, 5.5 mmol/L Mg^2＋,0.8 mmol/L dNTP mix,0.3 μmol/L each primer,0.2 U/μL rTaq,0.1 μmol/L probe and 1 μL template, and TaqMan PCR was carry out by the condition of 94℃5min→40 cycles（94℃ 15s→60℃ 45 s→read template）. Conclusion： A valid TaqMan PCR method was established, and by which the copy concentration of CYP3A29 and β-actin mRNA can be determined, so expression level of CYP3A29 mRNA in Bama miniature pig＇s tissues can be calculated.