论文部分内容阅读
目的:实时定量逆转录PCR(RT-q PCR)结果的标准化对于保证最终结果的准确性尤其重要。常用的标准化方法包括用加入的核酸量校正(ΔCt法)、用单个内参照基因校正(ΔΔCt法)和用统计学软件计算多个内参照基因的几何平均值进行校正。我们在db/db小鼠肝脏中对各种校正方法进行评估。方法:用ge Norm和NormFinder两种软件评估ACTB、e IF5、GAPDH、HMBS、HPRT1、Polr2A和RPLP0共7个内参照基因,以肝脏脂质合成相关基因Thrsp、SCD、SREBP1c和FAS作为目的基因。结果:应用ΔCt法,db/db小鼠肝脏中所有目的基因及GAPDH的表达显著升高(P<0.05)。ge Norm计算认为ACTB和HMBS最稳定。Norm Finder计算认为ACTB最稳定,而GAPDH和RPLP0为最佳组合。以单个基因ACTB或RPLP0,以及ACTB与HMBS,或GAPDH与RPLP0的几何平均值进行校正,db/db小鼠肝脏中除SREBP1c以外,Thrsp、SCD1和FAS的表达均升高(P<0.05)。结论:用ΔCt法校正RTq PCR的结果稳定并具有生物学意义。统计学软件ge Norm或Norm Finder应与ΔCt法整合使用。
Purpose: Standardization of real-time quantitative reverse transcription PCR (RT-q PCR) results is of particular importance to ensure the accuracy of the final result. Commonly used standardization methods include correction with the amount of added nucleic acid (ΔCt method), with single internal reference gene correction (ΔΔCt method) and with statistical software to calculate the geometric mean of multiple internal reference genes. We evaluated various calibration methods in db / db mouse livers. METHODS: Seven internal reference genes of ACTB, e IF5, GAPDH, HMBS, HPRT1, Polr2A and RPLP0 were evaluated by ge Norm and NormFinder software. Thrsp, SCD, SREBP1c and FAS genes related to liver lipid synthesis were used as target genes. Results: Using ΔCt method, the expression of all the target genes and GAPDH in db / db mice liver were significantly increased (P <0.05). ge Norm calculates that ACTB and HMBS are the most stable. Norm Finder calculates that ACTB is the most stable, while GAPDH and RPLP0 are the best combinations. The gene expressions of Thrsp, SCD1 and FAS in db / db mice liver were all increased (P <0.05) with the single gene ACTB or RPLP0 and the geometric mean of ACTB and HMBS or GAPDH and RPLP0. CONCLUSION: The results of calibrating RTq PCR using the ΔCt method are stable and of biological significance. The statistical software ge Norm or Norm Finder should be integrated with the ΔCt method.