Greetings,
I am currently working on a project that involves examining the differences in gene expression within groups. During the course of my research, I have discovered that the primers I am using amplify both cDNA and genomic DNA. This has led me to question whether this will impact my results. It is possible that the quantity of these genes is consistent in genomic DNA but different in mRNA, which means that the results may not be affected. Could you please advise me on this matter?
Thank you.
Hi Nour
Short answer: Yes, it's a problem if you have genomic DNA with your cDNA when you lauch your qPCR. You should remove the gDNA before the retrotranscription step.
If you're working with cDNA, it means you started out with RNA and had a retrotranscription step in your method. Your protocol should include a DNase digestion step before the retrotranscription. This is to eliminate genomic DNA from the sample. I reccomend you also run a gel at that point to see if the DNase treatment was complete. Sometimes, depending on the amount of gDNA, you may need to repeat the DNase step.
Since you're looking at gene expression by amplifying your target and comparing between samples, having gDNA in the mix means you'll be getting a signal not only from your cDNA (which serves as an indicator for your real expression) but also from your gDNA. The latter is a contaminant here, and a serious one because it's hard to quantify it's impact so you can't correct for it after the fact.
As for your statement :''It is possible that the quantity of these genes is consistent in genomic DNA but different in mRNA, which means that the results may not be affected'' , I'm not sure what it is you meant. However, you can't count on gDNA contamination being constant between samples, no matter how may copies of you target gene it's supposed to have.
I hope this helps, good luck!
Sandrine Toupin
Annica Gosch
Thank you for your clear and useful response. I appreciate your assistance
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