qPCR DNA template

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danushy
danushy's picture
qPCR DNA template

Hi, I am runnig qPCR to determine copy number of gene. I am using as template genomic DNA, and I´m doing standard curve with a plasmid that contains my gene of interest (absolute quantification).I have curves and products when I run the pcr in agarose gel. But when I calculte de r2 and the efficiency they are so wrong. The r2 is between 0.85-0.97 and the efficiency 122!!! Can anybody help me, because I don´t know which is my problem. In the melt curve there is only one peak, corresponding to the pcr product (it is correct). Thanks.
 

Ivan Delgado
Ivan Delgado's picture
Hi Danushy,

Hi Danushy,
Is there a reason why you are not using genomic DNA for your standard curve? Using plasmid DNA as your standard curve to quantify genomic DNA is not ideal (two very different templates).
As a rule of thumb an efficiency higher than 110% is indicative of inhibition (bad DNA, contamination in your reagents, etc). What kind of slope (from the linear regression of your curve) are you getting? 
This issue may well be associated with your low R-squared. R-squared is a measure of the variation in the DNA samples that make up your curve. In its simplest form, you simply did not pipet accurately. It it's worst form your assay is not good. 
Could you provide more details about your assay? How many dilutions are you running? Are these 10-fold dilutions? What is the concentration of your plasmid DNA? What are the Cts (too high, too low?)? Did you baseline corrected using automatic baseline or did you set the baseline by hand?

danushy
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Hi Ivan: thanks for your

Hi Ivan: thanks for your answer!!!
I am new in this and it is very difficult.
If i use genomic DNA for my stabdard curve, how would I know the number of copies (I mean i do 10 fold dilution for the plasmids and I know that the mass of plasmid i use corresponds to a certain copy number of plasmid) so if i use genomic DNA I only know the mass.
I run 5 dilution (10 fold) , beging with 10 pg of plasmid (1.39*106 copies). The Ct are for the 10 pg 12 and for the last dilution 0.001 pg (1.39 102 copies). The baseline I corrected using automatic baseline (I have I cycler )
There is no contamination in my reagents because there is no curve for the negative control (no template).
Again thank you for your answer !!!!!
 

Ivan Delgado
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I know how you feel. While

I know how you feel. While qPCR is pretty straight forward technique-wise, some of the mathematical calculations can be quite tricky.
Without going into too much detail, if you are working with human genomic DNA (genome size = 3.3 billion bp) then one genome is equal to 3.3 pg. If your gene of interest is found as a single copy in the genome, then there is one copy of your gene per 3.3 pg of genomic DNA. With that rationale, if you have 1 microgram (0.99 micrograns) of genomic DNA, then you have 300,000 copies of your gene. You can work out the other dilutions of your standard curve from that.  
Since you are getting your first Cts at 10-12, I would suggest playing around with the baseline by hand. Change the baseline from its default (typically cycles 3 through 15) and bring it down at least 1 or two Cts below the Ct of your first dilution (in this case, no higher than Ct of 9). Change this range of Cts (baseline) and every time see how the R-squared of your curve changes. This may help out. 
Let me know how either one of these approaches works out. 
Good luck

danushy
danushy's picture
Hi, me again with q PCR. I

Hi, me again with q PCR. I havea final question. I am working with CHO cells. The CHO cells genome size is 3*109 pb. According with your answer, 3 pg is 1 genome? Is this correct? Because I don´t know how to do the curve in copy number of genome.
Thanks in advance!!!!
 

Ivan Delgado
Ivan Delgado's picture
 

 
You are correct, a CHO genome (which is pretty much the same size as the mouse or human genomes) is ~3 billion bp long. That means that a haploid CHO genome weighs 3 pg, so if you have 9 pg you can say you have 3 copies of the CHO genome. The reason why in qPCR we think in terms of haploid genomes (3 pg) instead of diploid genomes (6 pg) is that one genome can only be copied once every PCR cycle (one copy yields two copies and not two strands create 2 new strands). Of course this is a fine line since you could argue that in "real terms" there is 6 pg of genomic DNA in one CHO cell since it is a diploid cell.
As a side: I recommend you not to quantify by qPCR below 10 genome copies. While it is technically possible (a CHO assay I designed a while back could quantify down to 0.1 CHO genomes) you will end up having problems (read about the Monte Carlo effect).
 
danushy wrote:

Hi, me again with q PCR. I havea final question. I am working with CHO cells. The CHO cells genome size is 3*109 pb. According with your answer, 3 pg is 1 genome? Is this correct? Because I don´t know how to do the curve in copy number of genome.
Thanks in advance!!!!