相对定量实验设计及条件优化.ppt

* 检测模式及应用 对照的设置 相对定量 如何选取看家基因 相对定量实验设置 实验评判标准 主要内容 * 相对定量实验的样本命名和类型设定 样本名 看家基因 （引物对1） 靶基因A （引物对2） 靶基因B （引物对3） Calibrator(对照组样本) 0 Reference Calibrator Target Calibrator Target Calibrator Negative(阴性对照) NTC Reference Negative Target Negative Target Negative Standard(标准品1) STD1 Reference Standard Target Standard Target Standard Standard(标准品2) STD2 Reference Standard Target Standard Target Standard Standard(标准品3) STD3 Reference Standard Target Standard Target Standard Standard(标准品4) STD4 Reference Standard Target Standard Target Standard Unknown(实验组1样本) 1 Reference Unknown Target Unknown Target Unknown Unknown(实验组2样本) 2 Reference Unknown Target Unknown Target Unknown Unknown(实验组3样本) 3 Reference Unknown Target Unknown Target Unknown Unknown(实验组4样本) 4 Reference Unknown Target Unknown Target Unknown Unknown(实验组5样本) 5 Reference Unknown Target Unknown Target Unknown Unknown(实验组6样本) 6 Reference Unknown Target Unknown Target Unknown Unknown(实验组7样本) 7 Reference Unknown Target Unknown Target Unknown * “10+10”的加样方案Primers+Taq Mix(5uL)=10uLDNA template+Taq Mix(5uL)=10uL Sample1 Sample2 Sample3 Sample4 Sample5 引物对A (Reference) 引物对B (Target1) 引物对C (Target2) 引物对D (Target3) 引物对E (Target4) * One to One: One target is paired with one reference according to the pipetting scheme, e.g., T1|R1, T2|R2 All to All: Each target is paired with each reference, e.g., T1|R1, T1|R2, T2|R1, T2|R2 All to Mean: Pairs each target with all references. The geometric mean of the resulting ratios is calculated, e.g., T1/R(all) = (T1/R1 x T1/R2)1/2 and T2/R(all) = (T2/R1 x T2/R2)1/2 Mean to All: Pairs all targets with each reference. The geometric mean of the resulting ratios is calculated, e.g., T(all)/R1 = (T1/R1 x T2/R1)1/2 and T(all)/R2 = (T1/R2 x T2/R2)1/2 LightCycler480 SW1.5 配对法则 * 检测模式及应用 对照的设置 相对定量 如何选取看家基因 相对定量实验设置 实验评判标准 主要内容 * 实验评判标准 特异性 Melting curve analysis for SYBR Green I assays Negative controls 灵敏度和动力学范围 Dilution series 效率 Standard curve 重复性