chip_pcr详细计算方法.docVIP

CHIP-SEQ?qpcr ?(2014-07-23 13:48:55)  HYPERLINK /s/javascript:; 转载▼ 标签：?  HYPERLINK /?c=blogq=%B9%C9%C6%B1by=tag \t /s/_blank 股票CHIP，用Q-PCR方法进行定量分析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、注意事项及实际应用：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行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因做两组标准曲线。Rotor-Gene?的会自动给出两组标准曲线的R值、扩增效率等信息，如果两组标准曲线的斜率，即M值的差小于0.1，表明两个基因的扩增效率已非常接近，那么后续实验中就可以用Comparative Delta-delta Ct法进行相对定量分析。反之，如果M差值大于0.1，就无法用该方法进行相对定量分析。此时的解决方法有两种，一是优化实验，使两组标准曲线的斜率差值小于0.1，二是换用其它的相对定量方法。双标准曲线法考虑到了不同基因扩增效率的差异，用标准曲线来校正扩增效率。双标准曲线法的特点、注意事项及实际应用：1.?双标准曲线法做相对定量分析的最大特点是，应用简便，无需像Del