I'm already done quantifying the viral DNA using absolute quantification (qRTPCR). Can anyone teach me how to convert my data into copies/gram? Currently it is in copies/ul.
Here are some information:
I used 50mg of tissue to extract the DNA in an extraction kit. After that, the extracted DNA were diluted by 10-2 . From this diluted DNA, I used 1 uL as DNA template in a 20uL reaction mixture of my qRTPCR.
Thanks!
Hi.
This is a bit tricky to calculate, because we need to understand that DNA extraction may not yield 100% of viral dna. But to compare the data, it is ideal to follow extraction protocol meticulously.... regarding the amount of each reagent taken at different steps including elution volume. If you have extracted your DNA evenly among the samples, then you can follow this method to calculate viral copy number per gm tissue.
if x is volume of extracted dna from 50 mg tissue
and y is copynumber per ul of 100 times diluted DNA
then your copynumber per gram tissue is 2000*x*y
(100*x*y/50/1000)
Hi Ramgie
You used 1uL of template, so your data tells you the copies per uL
Since this is a 1:100 dilution, you need to times this by 100 to give copies per uL for undiluted material
Next, you need to know the volume (in uL) of liquid you homogenised the tissue into - times by this number to give the total number of copies in 50mg
To convert this to copies per gram, simply times by 20
All the best
Thanks Barry Atkinson and Siva Ramakrishna Uppalapati for your responses.
Ok, I got it except for the last part wherein you multiply it by 20? I'm a little bit confused on where did you get this value?
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