Hi all
i want to analyze real time pcr results but i have no experience at all. My question is i have Cq value of my gene in resitant strain and suspectibile strain. Also i normalized them with reference gene and used control strain. i want to know gene expression difference between resistant and suspectible strain. i must calculate -ΔΔCq (Fold difference) or 2ΔΔCq (Relative quantity) ?
Best wishes
You have to calculate the fold difference to know how much is the difference between the resistant and susceptible strain (this will be the best information for showing your data). However, to know if this fold change is significant you have to do a t-test between both groups of the relative expression on your reference gene. So, in fact, you have to calculate both, one will offer you the statistical information, and the other will be the best for showing your results.
-ΔΔCq is a log fold difference, and or 2-ΔΔCq is a fold difference.
Standard statistical procedures work on the ΔCq values. The ΔΔCq is already the estimate of the mean difference. You can compare the ΔCq values between the two strains with a t-test (testing the hypothesis that the mean ΔCq values for both strains are equal, what is the same as testing if the mean difference of the ΔCq values between the strains is zero: ΔΔCp==0).
Hi simply u can use REST program ir u can use prism for gene expression using your ct values
Good luck
Thank you all for your help. i search some paper and as a result, i must use Livak aka ΔΔCp method for fold change between samples and calibrator (control) gene. My next goal is difference between resistant and suspectible strains. İn this scenario, i must normalize X gene expression for each sample( Resistance and suspectible strains? with the help of 2-ΔΔCq. After calculation, i must use t student test. i think its true?
Best wishes
The t-test is applied to the ΔCp values. You have two group of such ΔCp values: one for the resisstant strain and one for the susceptible strain. These two groups of ΔCp values can be used in a two-sample t-test.
Dear Jochen
in some of our Turkish-based articles, they are not using ΔCp. They are using
2-ΔΔCq(cq target-cq reference gene) and enter these values by using Mann-Whiyney U
That makes no sense. For such a comparison you calculate one single ΔΔCq value. You can not make a MW-U test on a single value. What could be is that they do the MW-U test on 2ΔCq values, what is possible but suboptimal.
My final question and i want to give an example
i have 3 resistance and 3 suspectible strain
These are ct values of each
Sample Resistance Target Reference ΔCq ΔΔCq(2-ΔCq)
1 17 15 2 0,25
2 16 14 3 0,125
3 21 14 7 0,007
Sample Suspectible Target Reference ΔCq ΔΔCq(2-ΔCq)
1 14 11 3 0,125
2 16 11 5 0,03
3 21 10 11 0,0004
Then, we enter these ΔΔCq values to SPSS or other software and gives us p value for difference or not?
What you call "ΔΔCq" are no ΔΔCq values. These are 2-ΔCq values.
The ΔΔCq value in your example is (3+5+11)/3 - (2+3+7)/3 = 2.33.
It is still smarter to "enter" the ΔCq and get the p-value based on the t-test.
You are right. ΔΔCq is not 2-ΔCq. But what is the mean of 2-ΔCq? is it the value of expression diference as a fold change?
No. 2-ΔCq is a kind of a "normalized expression value". It a measure for the expression of the target gene, normalized to the expression measure of the reference gene.It is a "fold-difference between the expression measure of the target and the reference gene".
The value itself is arbitrary and has no absolute meaning. It is not interpretable in isolation. However, such values can be compared between groups/conditions/treatments. Hence, the ratio of two "fold-differences" has the meaning of a "fold-change" (of the mormalized target gene expression between the compared groups.
i find a paper about this situation. Example 4 fits my research in my opinion. They use 2-ΔCq values and mean them. Then compare these groups.
Example 4 gives 50 fold difference. can we say that in tumor cells, BCL-2 expreses 50 fold higher than normal cells?
If the t-test gives you a p-value < 0.05 you can say that tumor cell express 50 times more Bcl-2 than normal cells. But if your t-test is not significant you cannot say that.
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