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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