Hi,
can you explain to me (in simple words) why i need to normalize my values after a RT-qPCR? In my case, I am studying how the expression of antibiotic resistance genes changes when the cells are grown in presence of the antibiotic. Now I have 2 RNA samples coming from cells grown in respectively an antibiotic-rich medium and an antibiotic-free medium. After RNA extraction, I had different amounts of RNA in the two different samples (measured at nanodrop) and I thought that this depends on the presence of the antibiotic in the medium..In my mind the fact that I have less RNA means that the expression of the gene will be less than normal..so I should see differences in the amplification with the real-time PCR, right? I was said that the normalization is done just because you might start with different concentrations of RNA in the samples and you want to eliminate the differences.but why should I eliminate the differences in RNA concentration between different samples if the difference in RNA concentration reflects a different gene expression?I did not understand why you need to use a housekeeping gene for this. Do you need to normalize also in case you start directly with DNA?I hope to have been clear.
Thank you so much
It is possible that your treatment could result in less RNA (or less cell growth and thus less RNA). You could determine if that is the case by doing replicates of your experiment and measuring RNA yield via the Nanodrop followed by a T-test. However, the reason for normalizing your expression to a housekeeping control gene is to correct your expression values for things like unequal starting material, unequal reverse transcription, unequal PCR reaction due to presence of a PCR inhibitor...In short, gene expression changes are almost always reported as a change in your measured gene relative to some other genes. In general, if one sample has twice as much total cDNA as another, you do not say that all of the genes are 2-fold increased.
Stating with DNA would be the same in most cases in that you would normalize your data to an internal control if possible.
Hi osric,
thank you very much for your answer. Can I ask you to explain me why "you do not say that all of the genes are 2-fold increased if the sample has twice as much total cDNA as another"? Maybe because you can have different types of RNA, not only the mRNA? Thank you so much..
I think this link is exact answer for your question.
http://www.nature.com/nprot/journal/v3/n6/abs/nprot.2008.73.html
Thomas D Schmittgen, Kenneth J Livak: Analyzing real-time PCR data by the comparative CT method.2008.
Silvia,
" if one sample has twice as much total cDNA as another, you do not say that all of the genes are 2-fold increased."
Gene expression has to be relative to something. A sample of cells that has half the cells of another sample and thus half the RNA certainly has differential growth but not necessarily differential gene expression.
The below article is the protocol for Analyzing real-time PCR data.
Thomas D Schmittgen, Kenneth J Livak: Analyzing real-time PCR data by the comparative CT method.2008.
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