Calculation of copy number for real time pcr
Posted 29 December 2020 - 10:58 PM
I would like to develop real time pcr assay to determine if there is any adulteration of pork in beef samples. However, I am not sure how to estimate the copy number of the target gene. While I would be using a single copy gene in nuclear genome, which is a beta actin, the reference gene would be myostatin, which is also a single copy gene. So in order to calculate the copy number of target gene (beta-actin), is my following calculation correct?
Based on NCBI genome, pork genome size is 2458 Mb
For a gDNA extracted from muscle tissue, say 100 ng,
Copy number = 100 ng * 6.022 x10^23 / ( 2458 Mb x 660 g) ?
Or I need to further divide the value by two as it?s a diploid genome?
Thank you very much for your help!
Posted 03 January 2021 - 02:36 AM
In qPCR you do this by doing absolute quantification.
You can do it relatively, but you will only get fold-change (using standard equations for target and reference gene). That still may be enough for your use though, just set a limit (max 1.1 fold of pork to call it adulterated) I can image it is not needed to be a copy number precise, but that is just my opinion.
To have absolute values, you need to take your gene of intererest and make it into plasmid, and copy that in bacteria. Plasmid is then measured and copy number of it calculated, in plasmid you know the exact lenght and have reliable concentrations ( I made a site for this, since I needed it: http://stanice.euweb.../DNAtoCopy.html )
In higly purified plasmid prep, this should be pretty precise. (but possibly linearize it first with a single cuting enzyme and purify, before measurement).
The linearized plasmid mixed with some dummy DNA like salmon-sperm DNA (to increase complexity enought to simulate gDNA) and diluted acts as standard for measuring copy number by absolute quantification.
You do this for the myostatin too. you than have two absolute vales that you only divide. Diploidy should be taken into account in both, but since it is in both you may just forget about it, since you divide those values anyway.
But as for beta-actin... it is not a favorable gene (contrary to popularity) for either the references, since it is single-copy but has many pseudogenes. If you are working with DNA, you can pick just anything, and I would say there would be more reliable single-copy genes than beta-actin.
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