I wonder if any one know how to obtain numbers of genome equivalent per ng of DNA from qPCR Ct values .
Thanks
I've previously done this by comparing to a plasmid standard curve. If you have your target sequence on a plasmid, you can then dilute that plasmid standard down to a range of 10 - 100,000 copies/uL. You can then estimate copy number from the Ct values you obtain from your genomic template. If you know the copy number of your gene per genome, you can then deduce how many genome equivalents are in a sample.
A key assumption here is that your primer set can amplify from genomic DNA as efficiently as it can from plasmid DNA, so it is important to test this assumption before you try to use it for real. Another caveat is that in normal diploid cells we know most genes have 2 copies but there are exceptions and, if you are working in cancer cell lines, you really can't assume that there are 2 copies per gene anymore.
I don't know if this is the best way to do it but it's worked for me in the past. If you're not totally committed to a qPCR based method, I think this might be easier to do with a NanoString assay.
I've previously done this by comparing to a plasmid standard curve. If you have your target sequence on a plasmid, you can then dilute that plasmid standard down to a range of 10 - 100,000 copies/uL. You can then estimate copy number from the Ct values you obtain from your genomic template. If you know the copy number of your gene per genome, you can then deduce how many genome equivalents are in a sample.
A key assumption here is that your primer set can amplify from genomic DNA as efficiently as it can from plasmid DNA, so it is important to test this assumption before you try to use it for real. Another caveat is that in normal diploid cells we know most genes have 2 copies but there are exceptions and, if you are working in cancer cell lines, you really can't assume that there are 2 copies per gene anymore.
I don't know if this is the best way to do it but it's worked for me in the past. If you're not totally committed to a qPCR based method, I think this might be easier to do with a NanoString assay.
Good advice from Govinda. One additional note: you can - you should! - nick or better cut (linearize) the plasmid before using it as template in the qPCR.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC156059/
Shortcut for lazy boys, but don't trust it too much. You have to adjust it for your method, anyway. Decent method is exactly as Godvinda wrote, with calibration of your plasmid dilutions with external qualitiative reference material.
@Govinda Sharma I know this post is 2 years now. However i am experiencing similar problem. I am trying to quantify the the number of bacteria in my milk samples. I want to report this answer in genome equivalents. As stated above i have diluted my standards and generated a standard curve. I have the ct values of all my standard dilutions and my samples as well. However i do not know what to do with these values in order to get the genome equivalents of DNA in my samples. My questions are 1. How do i estimate the copy number from ct values?, also how can genome equivalents be deduced from these copy numbers? Many thanks
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