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Calculation of virustiter after qPCR


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#1 LabWorker123

LabWorker123

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Posted 14 October 2013 - 06:54 AM

Hello everyone,

 

I want to define LCMV virus titers in different organs/serum. Therefore I weight the organs, isolated RNA (elution with 60ul ddH2O), reverse transcribed 1ul RNA in a 20ul total approach and run qPCR with 10ul cDNA diluted 1:10 with H2O in 25ul total volume. Next I used a appropiate DNA plasmid standard curve to calculate the virus copies.

 

Now I want to calculate the virus copies/miligramm of organ - how do I have to insert the dilutions from elution/dilution into my calculation?

 

My suggestion is: if I have x copies according to the standardcurve I would multiplicate them with 60 (because I took 1ul of 60ul eluate for reverse transcription) multiplicate with 10 (dilution before qPCR) and divide through the initial organ mass e.g. I have 300 copies from 25mg of liver => (300X60X10)/25 = 180.000 copies/mg

 

Thank you for your help

 

LL






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