Dear All,
We work on a project with aims looking at the changes brought about by ketogenic diet( a special form of diet found to be effective in treating drug-resistant epilepsies). We have collected stool samples of the patients at two times points ( 1. baseline- before the initiation of ketogenic diet 2. one year after the diet therapy initiation). DNA has been extracted from the stool samples by Qiagen kit method and quantified by nanodrop method. The DNA samples were then diluted to 1 ng/uL. Real-time PCR were carried for these samples using primers and probes specific for Bacteroidetes, Prevotella and Firmicutes. We have thus obtained Ct values for each of the samples , for all the three bacterial groups.
We have loaded the same amount of template DNA for each reaction (1 ng). We would like to do relative quantification (fold increase). Is it wrong to compute the fold increase by the following method?
For example, if Ct (baseline)= 20 & Ct (one year) = 19
Ct(one year)- Ct(baseline) = 20-19 = 1
Fold increase = 2^1 = 2
Please let us know.
Thank you in advance
It should be Ct(one year) - Ct(baseline) i.e., = 19-20 = -1 and then you can calculate fold change using formula in excel i.e., =POWER(number, -power). In this case it will be =POWER(2, -(-1)) = power(2, 1) = 2. Which explains that relative to baseline you have 2 fold increase in expression.
Hi Magith,
Have a look on the attached paper. You will get an idea from this link ( https://bitesizebio.com/24894/4-easy-steps-to-analyze-your-qpcr-data-using-double-delta-ct-analysis/ ) also.
Best wishes.
Hi Magith. I go with the reply of Ritesh. It is the correct way. I would like to know if you had taken some internal controls; in your case, some bacterial strain that does not get affected by the keto-diet. I feel this is important to rule out any changes in the gut bacteria, over a period of one year, due to various uncontrolled physical and physiological factors; hence lowering your false positive or false negatives.
Good luck.
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