1. my reference gene is beta-actin, Shall i go with absolute quantification or comparative ct method as i do not have treated and untreated samples????
2. can i do the Real time PCR reaction for both my gene and beta actine in the same tume or different tubes???
sorry for taking much of your time, but i am still new in the field of real time PCR. Thanks in advanced for your valued help.
I can not understand your question exactly.
Use ct and run different tubes.
Thank you Dr Alireza, to make it clear.
i need to know only the expression level of my gene of interest in two cell lines. As i do not have treated and non treated samples i realized that i can't use the comparative quantitative method or (delta delta ct). Also, i need to normalize my gene of ineterest gene expression level by using beta actin as my reference gene for each cell line ( please correct me if iam wrong). To do that, by only running real time PCR i will be having two ct(s) for each cell line prepared in two different reactions, one for my gene of interest and the other for my reference gene.
can i determine the fold expression of my gene of interest only by dividing the ct of my gene of interest over the ct of my reference gene ???? or this is wrong??? if this is wrong how can i determine it???
When you use housekeeping gene(reference gene), delta ct shows you change of expression your target gene.
Delta delta ct also can be used without any treatment for example times when change expression of your first target gene changing expression your second target, but you have tow different cell lines.
Do you mean i compare them to each other by delta delta ct , i can't get that clearly Dr Alireza???
That was an example dear Balsam.
I said that if you monitoring two genes expression in one cell line and each other of them can affect expression other you can use delta delta ct.
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