What Real Time-PCR data analysis method must I use? - (Feb/28/2008 )
Hi everybody,
I would like to perform a Real Time PCR with some genes I'm studing before a treatment with a inhibitor and I'm plenty of doubts:
1- What Real Time analysis data method must I use?. I want to compare the expression levels of my genes before and after the treatment.
2- Would be necessary to include a standar curve?. How would you perform it?.
Thank you in advance for your help.
Hi there!
I would suggest the delta-delta ct method. By comparing a housekeeping gene with your gene of interest, you will be able to see a relative variation of your gene after the treatment.
As for the standard curve, you will have to do it before your delta delta ct experiment. The purpose of this curve is to make sure that the PCR reactions for the two pairs of primer (housekeeping and your gene) have the same efficiency. I order to do it, you have to make serial dilution (1, 0.1, 0.01, 0.001 etc). and then perform the PCR reaction.
I would suggest the delta-delta ct method. By comparing a housekeeping gene with your gene of interest, you will be able to see a relative variation of your gene after the treatment.
As for the standard curve, you will have to do it before your delta delta ct experiment. The purpose of this curve is to make sure that the PCR reactions for the two pairs of primer (housekeeping and your gene) have the same efficiency. I order to do it, you have to make serial dilution (1, 0.1, 0.01, 0.001 etc). and then perform the PCR reaction.
I have had a similar question. And a few more. As I am a newbee in qRT-PCR and I `ve actually read some info about statistical analysis and interpreting the data, what I consider the most important, and unfortunatelly most difficult.
Could someone please to say it simply and explain those:
1. how to find delta delta Ct. For example I have Ct for my gene 18.4 and for control gene 20.2 what is quite close to my results.
2. Ct is the only thing I can get from qRT-PCR facility comp ( not counting Print Screen images) and then I have delta delta Ct
3. How it is better to show results on seminar or ie. in a paper. Many people doing it in many different ways. What you would suggest the best possible way to do it?
4. How can I transform it to the level of gene expression? I know that without right controls you don`t have absolute level of gene expression but relative. And can you at least approximately say by how many times your gene of interest is lower expressed or higher then control?
I know it may seem that questions are to easy and I should find it by myself, but I and I think many aothers would appreciate simple and easy to understand answer
Many thanks,
Andy
Nice article (with examples) about analysis of relative gene expression data using real time quantitative PCR and the delta delta Ct method
[attachment=4327:Livak___...hod_2001.pdf]
[attachment=4327:Livak___...hod_2001.pdf]
Many thanx!!!!! I will look through it ..
Thanks for your help!!