Hello All,
I have several questions about qPCR. I will explain what I have done to give you a clear picture.
I have 2 kidney samples. 1 wildtype and 1 KO for a specific gene.
We have extracted genomic DNA from both samples.
I then want to evaluate the effectiveness of the KO. So, I designed 2 primers: 1 within the region between the 2 loxp sites "the region that is supposed to be deleted in the KO samples". And the other primer amplyfing the loxp site.
I then made a 2-fold serial dilution of the samples, and ran a qPCR using the serial dilutions of both samples "control and KO" and using the primers I have designed + 2 reference "housekeeping" genes.
Just to remind you that this is genomic DNA not cDNA.
My questions are:
First of all is my protocol and the steps I have done right? or do you have any suggestions of modification?
What is the best way to analyze the results? is relative quantification possible in gDNA qPCR (delta delta Ct or pfaffle method) ? or only absolute quantification is possible (standard curve)?
Please help ...
You don't need to use qPCR to answer this question. Design primers that would differentiate between the WT and KO alleles and see if your cells are heterozygous, homozygous WT, or homozygous KO. Include a full set of controls.
You pointed out an important consideration regarding using qPCR with genomic DNA. Indeed, ΔΔCt method can be applied to gDNA for relative quantification, contrary to a common misconception that it's only suitable for cDNA or expression analysis. The key here is the selection of a reference gene. Contrary to selecting a housekeeping gene based on its constant expression, for gDNA, the emphasis should be on choosing a 'single-copy gene' that maintains a constant copy number in each cell.
As for the actual protocol, I don't have one, as my personal experience with this method is specifically using it for the relative quantification of telomere length. That being said, I believe that it could work for your case. By choosing a good single-copy gene as your reference, you can assess the relative abundance of the target sequence (the region between the two loxP sites or the loxP site itself) in the KO vs WT samples.
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