I have calculated 2 (-ddct) for a set of 6 genes in 5 tissues using GAPDH as reference and one of the tissues as the calibrator. However, after computing the 2 (-ddct) values, which I believe is same as the fold change (please correct me if I am wrong), I was told that relative expression is not same as fold change [2 (-ddct)]. If so, what is the difference between fold change and relative expression ?
To understand the concept of gene expression using real-time PCR, one should be familiar with the terms:- (1). Absolute gene expression (2 -dcT) (2). Relative gene expression ( 2 -ddcT) (3). Test gene (4). Housekeeping gene (5). Test sample (6). Control sample.
Case I- Suppose you want to assess the expression of gene x in your test samples (in vivo, in vitro). You need to get ct values for test gene x and housekeeping gene (HK). You will normalize gene x from HK gene this will give you delt cT value and calculate it as 2 -dcT. This will give you the absolute expression of gene x after treatment i.e. how much is the level of gene x after treatment in test samples.
Case-II- Suppose you want to compare the level of expression of gene x (after treatment) with control set. Here you need to get cT values for test gene x and HK gene in test samples as well as in control samples. First, normalize the cT values with housekeeping for test sample as well as control sample i.e. dcT. Now normalize the dcT of the test sample with the dcT of the control sample, this will give you ddcT value, calculate it as 2 -ddcT. This is the relative gene expression i.e. what is the level of expression of gene x in test samples in comparison with control samples. The 2 -ddcT of control samples is always 1 (negate dcT of control set with itself, you will get 0 and log base 2 of 0 is 1). So if your value is more than 1, expression of gene x is increased after treatment and the value is less than 1, the expression is decreased after the treatment in comparison to the control set.
For more clarity of the concept, you can go through the following papers
*Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3, 1101–1108
*Livak KJ, Schmittgen TD. 2001. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25, 402–408.
Hope this will help.
First of all, for RT-qPCR results, the relative expression level of each gene needs to be normalized by two housekeeping genes (and not on GAPDH only); as recommended by the MIQE guidelines (Bustin et al., 2009).
Then, for the fold-change, the relative expression and the 2^(-ΔΔCt)...they are not the same thing (even if they are finely linked).
After computing your RT-qPCR slopes & curves data you obtain your 2^(-ΔΔCt) data. During this procedure, you have to normalise all your data on housekeeping genes (a couple) and on a "control experiment" (associated as the "1:1" value) to compare them as ratio-values: data are presented as relative expression of your genes. --Be careful to use geometric means at this step!--
For me, the fold-change is the factor between data (e.g.: "the expression of Bcl2 in the experiment A is 40-fold higher than in the experiment B").
If you want more more precise and detailed informations, please see Livak and Schmittgen (2001).
Dear community of RG, be free to add modifications & precisions to my Answer!
To understand the concept of gene expression using real-time PCR, one should be familiar with the terms:- (1). Absolute gene expression (2 -dcT) (2). Relative gene expression ( 2 -ddcT) (3). Test gene (4). Housekeeping gene (5). Test sample (6). Control sample.
Case I- Suppose you want to assess the expression of gene x in your test samples (in vivo, in vitro). You need to get ct values for test gene x and housekeeping gene (HK). You will normalize gene x from HK gene this will give you delt cT value and calculate it as 2 -dcT. This will give you the absolute expression of gene x after treatment i.e. how much is the level of gene x after treatment in test samples.
Case-II- Suppose you want to compare the level of expression of gene x (after treatment) with control set. Here you need to get cT values for test gene x and HK gene in test samples as well as in control samples. First, normalize the cT values with housekeeping for test sample as well as control sample i.e. dcT. Now normalize the dcT of the test sample with the dcT of the control sample, this will give you ddcT value, calculate it as 2 -ddcT. This is the relative gene expression i.e. what is the level of expression of gene x in test samples in comparison with control samples. The 2 -ddcT of control samples is always 1 (negate dcT of control set with itself, you will get 0 and log base 2 of 0 is 1). So if your value is more than 1, expression of gene x is increased after treatment and the value is less than 1, the expression is decreased after the treatment in comparison to the control set.
For more clarity of the concept, you can go through the following papers
*Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3, 1101–1108
*Livak KJ, Schmittgen TD. 2001. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25, 402–408.
Hope this will help.
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