It would be more convenient if someone could suggest some excel sheet method/software to calculate delta ct and folds expression. Manually one by one is quite complex and time consuming.
If you've calculated your deltaCts, you've established the difference in Ct between your test TARGET(S) (e.g. a gene targeted by siRNA) and your control TARGET (usually a housekeeping gene, e.g. GAPDH)
To go from deltaCts to quantitation in excel, you first need to calculate the deltadeltaCts; this is the deltaCt of your test SAMPLE(S) (e.g. a knockdown sample) minus the deltaCt of your control SAMPLE (e.g. a scrambled control treatment).
The final step is quantitation, which will give you the amount of test TARGET in your test SAMPLE relative to the amount of test TARGET in your control SAMPLE. The formula for this is: quantitation = 2^(-deltadeltaCt).
So, for example, consider the following example:
Scrambled control: GAPDH=18, HC11=26
Knockdown treatment: GAPDH=19, HC11=29
Now, the deltaCts are as follows:
Scrambled control: 26-18 = 8
Knockdown treatment: 29-19 = 10
This means that the delta delta Ct is 10-8 = 2
Finally, we quantify: 2^(-2) = 0.25
This means there is 25% of the HC11 in the test sample as the Scrambled control, suggesting that the knockdown has resulted in a 75% decrease in HC11 levels.
I hope this helps!
If you've calculated your deltaCts, you've established the difference in Ct between your test TARGET(S) (e.g. a gene targeted by siRNA) and your control TARGET (usually a housekeeping gene, e.g. GAPDH)
To go from deltaCts to quantitation in excel, you first need to calculate the deltadeltaCts; this is the deltaCt of your test SAMPLE(S) (e.g. a knockdown sample) minus the deltaCt of your control SAMPLE (e.g. a scrambled control treatment).
The final step is quantitation, which will give you the amount of test TARGET in your test SAMPLE relative to the amount of test TARGET in your control SAMPLE. The formula for this is: quantitation = 2^(-deltadeltaCt).
So, for example, consider the following example:
Scrambled control: GAPDH=18, HC11=26
Knockdown treatment: GAPDH=19, HC11=29
Now, the deltaCts are as follows:
Scrambled control: 26-18 = 8
Knockdown treatment: 29-19 = 10
This means that the delta delta Ct is 10-8 = 2
Finally, we quantify: 2^(-2) = 0.25
This means there is 25% of the HC11 in the test sample as the Scrambled control, suggesting that the knockdown has resulted in a 75% decrease in HC11 levels.
I hope this helps!
@Daniel Scott thanx but i know this method ,known as Livak method but since i have more than 500 samples so i want to know about some online free software ,that makes it easy,,,,,
Ah, fair enough - that would be tedious! I'm afraid I've not used any free online software as most of my datasets are smaller.
If you did want to do it in Excel, and provided that your experimental outputs are pretty uniform in layout (e.g. you're always putting the same kinds of samples in the same wells etc.) you could always write a couple of macros to automate the analysis for you? If you could do that, you'd only ever have to do the long analysis once to write the macro then you could just replicate it with one keyboard shortcut? Not sure if your samples are laid out to allow that, but just a thought!
Good luck!
hi..write the formula in excell and it will calculate the fold change quickly. then you can analyze it easily by prism software
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