Can someone please suggest online free software for the analysis of relative gene expression after obtaining delta ct values on real time pcr?

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!

the REST software is NOT compatible with x64 Windows 8.

hi..write the formula in excell and it will calculate the fold change quickly. then you can analyze it easily by prism software

I know it's been 8 years since you posted this but I wrote a python program for delta delta ct analysis all you have to do is enter the Cq/Ct values manually and it will do the rest of the math for you. Here is the link to the program and the directions on how to use https://github.com/jfshaffe/qRT-PCR-Calculator

In my experience it cuts analysis time approximately in half. The program assumes you are using three technical replicates per condition and one experimental sample/one control sample/one experimental gene/one housekeeping gene

u may use Excel

I suggest this excellent online tool which gives accurate calculations. You can use it to calculate gene expression according to the Liavk method for example. The link is