均匀设计在候选内参基因RT-qPCR实验条件优化中的应用

摘要

Objective To investigate the application of the uniform design to optimize quantitative real-time RT-PCR conditions, and apply it for assessment of potential internal control genes in THP-1 cells under different conditions. Methods We obtained from cDNA THP-1 cells. The cDNA template quantity, primer quantity and annealing temperature were studied as 3 key factors for the RT-qPCR conditions. By using the uniform design, 8 levels for each factor were optimized. The standard of score and a standard curve of 5-fold dilution series (1 : 101 ,11 102,1:103,1:104 and 1:105) of template cDNA were used to estimate the amplification condition. Results By using the uniform design, RT-qPCR conditions for assessment of potential internal control genes were obtained from 8 tests in one experiment. The optimized conditions were cDNA template concentration of 0. 5μg, each primer concentration of 200μmol/L in the final reaction, and annealing temperature at 55℃. Conclusion The uniform design is a highly efficient method to optimize the RT-qPCR conditions for potential internal control genes in this study. Furthermore, the uniform design is suitable for the multi-factor and multi-level test conditions.%目的 探讨均匀设计在候选内参基因RT-qPCR实验条件优化中的应用,有助于后期实验筛选不同状态下THP-1细胞的稳定内参基因.方法 以THP 1细胞cDNA为模板,cDNA模板量、引物浓度和退火温度3个因素为影响因素,应用均匀设计3因素8水平,探索影响候选内参基因RT-qPCR的扩增条件,检测不同实验条件下的扩增效果,在最优扩增条件下建立候选内参基因定量扩增的相对标准曲线,并检测扩增效率.结果 通过均匀设计,完成对cDNA模板量、引物浓度和退火温度3因素8水平的实验条件优化,建立候选内参基因RT-qPCR检测的最佳组合为cDNA模板量0.5μg、引物浓度200μmol/L、退火温度55℃.结论 本实验选用均匀设计优化RT-qPCR实验条件,筛选出THP 1细胞候选内参基因RT-qPCR的最优实验条件.均匀设计适合于本实验研究多因素多水平试验条件的配比.