加载中…简单五步学会使用∆∆Cq法(∆∆Ct法)计算实时定量PCR(qPCR)基因表达差异
(2014-06-24 16:14:45)
标签：
pcr表达差异统计双deltact法双deltacq法实时定量pcr定量pcr结果统计 |
分类： 分子与细胞生物学 |
用一个简单的例子，旨在演示某种处理(treatment)下某基因(TAR)的mRNA水平变化（为了简化，假设这次实验每组6个样，即生物重复，没有使用技术重复，并假定其内参基因(REF)稳定，不再涉及内参的可靠性等问题）
即只简单演示∆∆Cq法如何计算倍数改变。
主要分为如下五步：
第一步：经非参基因进行归一化(Normalize to REF): ∆Cq=Cq(TAR)-Cq(REF)
第二步：转化为指数表达(Exponential expression transform)：∆Cq Expression=2-∆Cq
第三步：组内样品指数表达的平均值与标准差(Average replicates and calculate standard deviation)
第四步：以对照组为参照进行归一化(Normalize to treatment control)
第五步：变化的百分比[%change=(1-∆∆Cq)*100]
|
A |
B |
C |
D |
E |
F |
G |
H |
T |
J |
|
Groups |
Cq FER |
Cq TAR |
∆Cq |
∆Cq Expression |
Mean ∆Cq Expression |
∆Cq Expression Std. Dev. |
∆∆Cq Expression |
∆∆Cq Expression Std. Dev. |
% change |
|
|
|
|
=CqTAR-CqREF |
=2^-Δ∆Cq |
Average Replicates |
Std. Dev. Replicates |
Normalized to mean in control |
Normalized to mean in control |
=(1- ∆∆Cq)*100 |
|
Treatment |
20.6 |
27.6 |
7.0 |
0.0078 |
0.0215 |
0.0133 |
0.0639 |
0.0395 |
93.61 |
|
|
20.8 |
27.3 |
6.5 |
0.0110 |
|
|
|
|
|
|
|
20.9 |
27.6 |
6.7 |
0.0096 |
|
|
|
|
|
|
|
20.7 |
25.6 |
4.9 |
0.0335 |
|
|
|
|
|
|
|
20.6 |
25.4 |
4.8 |
0.0359 |
|
|
|
|
|
|
|
20.6 |
25.6 |
5.0 |
0.0313 |
|
|
|
|
|
|
Control |
20.5 |
22.2 |
1.7 |
0.3078 |
0.3369 |
0.0707 |
1.0000 |
0.2098 |
|
|
|
21.2 |
22.5 |
1.3 |
0.4061 |
|
|
|
|
|
|
|
21.0 |
22.5 |
1.5 |
0.3536 |
|
|
|
|
|
|
|
21.2 |
22.5 |
1.3 |
0.4061 |
|
|
|
|
|
|
|
21.0 |
22.6 |
1.6 |
0.3299 |
|
|
|
|
|
|
|
21.2 |
23.4 |
2.2 |
0.2176 |
|
|
|
|
|
各栏目的含义如下：
Column A: 分组
Column B: 参考基因的Cq值(Cq value for REF)
Column C: 靶基因的Cq值(Cq value for TAR)
Column D: 归一化到相应参考基因的表达(Normalize Cq values for all TAR samples to the REF gene of its corresponding sample, ∆Cq)
Column E: 指数转化，该方法的前体是100%的扩增效率(Exponentially transform ∆Cq to ∆Cq Expression for each biological replicate; 2 raised to the -∆Cq yields ∆Cq Expression. 100% qPCR amplification efficiency for all reactions, or a doubling of amplicon with each subsequent qPCR cycle.
Column F: 指数转化值的组内均值(Mean of ∆Cq Expression replicates.)
Column G: 指数转化值的组内标准差(Standard deviation of the mean for ∆Cq Expression replicates.)
Column H: 与对照组归一化处理，获得∆∆Cq表达值(Normalize the TAR Mean ∆Cq Expression to that of the Control to obtain ∆∆Cq Expression.)
Column I: ∆∆Cq表达值的标准差(To find the standard deviation of ∆∆Cq Expression, divide the standard deviation of the targeted sample’s Mean ∆Cq Expression by that of the Control sample.)
Column J: 处理所致变化的百分比(Percent change is calculated by subtracting the normalized ∆∆Cq Expression from 1 (defined by the level of expression for untreated sample) and multiplying by 100.)
参考文献
Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001, 25, 402–408.
Thermo. Demonstration of a ∆∆Cq calculation method to compute thermo scientific relative gene expression from qPCR data
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