Hi all,
Can anybody suggest me the steps required to perform quantitative real-time PCR analysis of mRNA expression data. Actually, I have some differentially expressed genes and I want to validate them by real-time qPCR but I don't have any idea, how to perform real-time qPCR. I have searched the web in this regard but not able to find any suitable answer. If someone please suggest me some good paper to understand that or provide some link to perform this process online then it will be very helpful for me. I am from the computer science background, working in the computation analysis of mRNA data.
The most important question is if you have wet-lab experience. Even though qPCR is not rocket science at all, the RNA isolation requires some hands-on experience to avoid RNase contamination, and pipetting a 96-well should be practiced before running your experiment, otherwise it will most likely be a mess. Get in contact with an experienced colleague to assist you, you'll not learn this overnight without any pre-experience.
Usually qPCR machines will produce Ct values and you'll want to use the "delta delta Ct" method to look at differences between group, where the fold-change is 2^-ddCt (ddCt is the delta delta Ct). So the steps are:
Subtract the Ct for your gene of interest from the internal control in each sample. This is the "delta Ct".
For each group, take the average of above
Subtract the average delta Ct values, produce the ddCt.
Compute the fold-change as indicated above.
A p-value can be computed with a T-test or Mann-Whitney U test from the "delta Ct" values.
Regarding actually performing qPCR, order some TAQman primers and read the instructions, it's pretty simple. You'll need an appropriate machine, but they're pretty trivial to find.
Dear Mr. Ryan,
Thanks for your informative and quick response. But, I have some queries regarding differentially expressed gene analysis. I have identified some differentially expressed gene by my computation method. Will you suggest me some real validation method, (Except pathways & GO analysis) so that I can validate the importance of these identified genes. As, I am not from a biology background, hence having some problem to identify an exact solution. will real-time PCR analysis serve the purpose?
Once again thanks for your valuable reply.
Regards
Shib Sankar
Real-time PCR validates that the change you see is real in a larger number of additional samples. Whether a change is actually important requires you to understand the underlying biology. You should spend a few days reading the literature related to the research project.
The most important question is if you have wet-lab experience. Even though qPCR is not rocket science at all, the RNA isolation requires some hands-on experience to avoid RNase contamination, and pipetting a 96-well should be practiced before running your experiment, otherwise it will most likely be a mess. Get in contact with an experienced colleague to assist you, you'll not learn this overnight without any pre-experience.