Can I use genomic DNA to make a standard curve for qPCR - (Dec/17/2009 )
I am going to do qPCR for cDNA from E. coli. I learned that I should do standard curve for each set of primers on every plate. But I don't have enough cDNA of one sample that I can use for standard curve for all the rest of my experiments (maybe ten more plates and around ten sets of primers). Can I use genomic DNA to do the standard curve? Thank you for your help!
catheriney on Dec 17 2009, 08:42 PM said:
No you can't use the genomic DNA to get a standard curve. You will get false positive results as I have tried once. The other option I used was 18s rRNA as a control as well to quantify my qPCR. But since I used for the mammalian cells you can try to look out for the eukaryotic equivalent of 18s rRNA.
Thank you, sameer. What do you mean false positive results?
I thought that since genomic DNA contains all genes I want, it should be ok for me to use it as the template to make the standard curve. Is it right?
As for the internal control, I do use 16S rRNA.
sameermahmood on Dec 21 2009, 11:01 AM said:
imho you can use genomic dna as standard. it is not relevant where your dna came from for the standard curve, it is just important that the correct target sequence is contained in your standard and that the dilutions are accurate. You can also use plasmid, cDNA, PCR products, ...
an exception is an absolute standard curve if you are quantifiying mRNA, then you should also use reverse transcribed RNA (eg. IVT RNA) for your standard curve.
I agree with tea-test, with the following proviso - genomic dna often has high molecular weight, and it can be challenging to get good reliable dilutions of it without first shearing or otherwise reducing its stringiness. Shearing first by expression through fine needles or partial digestion with a frequent cutter would make this less of a problem.
I think you CAN use genomic DNA for qPCR, but you need to sheer it into smaller fragment.
And also, unless you are very sure that the gene you want to amplify has only ONE copy per genome., Otherwise it is not a good idea to use genomic DNA.