chip-pcr-详细计算方法VIP专享

CHIP-SEQ qpcr (2024-07-23 13:48:55)▼标签： 股票CHIP，用 Q-PCR 方法进展定量分析i. Normalizeeach ChIP DNA fractions’ Ct value to the Input DNA fraction Ct valuefor the same qPCR Assay (ΔCt) to account forchromatin sample preparationdifferences.ΔCt [normalized ChIP] = (Ct [ChIP] - (Ct [Input] -Log2 (Input Dilution Factor)))Where, Input Dilution Factor = (fraction of theinput chromatin saved)-1Average normalized ChIP Ct values for replicate samples.ii. Calculate the % Input for each ChIP fraction(linear conversion of the normalizedChIP ΔCt).% Input = 2 (-ΔCt [normalized ChIP])iii. Adjust the normalized ChIP fraction Ct valuefor the normalized background(NIS/mock IP) fraction Ct value (first ΔΔCt).ΔΔCt [ChIP/NIS] = ΔCt [normalized ChIP] - ΔCt[normalized NIS/mock]iv. Calculate Assay Site IP Fold Enrichment abovethe sample specific background(linear conversion of the first ΔΔCt).Fold Enrichment = 2 (-ΔΔCt [ChIP/NIS])v. Determine the difference between the normalizedexperimental sample (S2) andthe control sample (S1) ChIP fraction Ct values(second ΔΔCt).ΔΔCt [S2-S1] = ΔCt [S2:normalized ChIP] - ΔCt[S1:normalized ChIP]vi. Calculate Differential Occupancy Fold Change(linear conversion of the secondΔΔCt to yield a fold change in site occupancy).Fold Change in Occupancy = 2 (-ΔΔCt [S2-S1])看到有两种 ChIP 完定量的计算方法：1〕双△CT 法；2〕双标准曲线法。Comparative Delta-delta Ct 法的特点、考前须知与实际应用：1. Comparative Delta-delta Ct 法的一大特点是，当优化的体系已经建立后，在每次实验中无需再对看家基因和目的基因做标准曲线，而只需对待测样品分别进展 PCR 扩增即可。2. 每次实验都默认目的基因和看家基因的扩增效率一致，而并非真实扩增情况的反映，因此实验条件需要严格优化，并且总会存一定的偏差。用 ComparativeDelta-delta Ct 法展开定量实验前，在预实验中，必需对目的基因和看家基...