Hi all,
I'm going to be generating an RT-qPCR standard curve to quantify genome copies as a proxy for PFU in a superinfection experiment I'm going to be doing (so I can't just do a plaque assay because there will be 2 viruses present). I was planning to use a stock of virus that I have, and I was thinking I'd like to aim to start with about 100-200 ng RNA worth of virus to extract, make a cDNA library, and run qPCR on it. I was trying to do the math to figure out how much of my stock I'll need, and it seems like I'm off somewhere... here's what I've got (back of the envelope):
genome MW:
= ~12kb ssRNA virus x 340 g/mole (average ribonucleotide MW)
= 4E6 g/mole genome copies
= 4E6 g/6E23 genome copies
= 4E15 ng/6E23 genome copies
= 6.6E-9 ng/genome copy (eyeballing, this still seems reasonable)
= 1.5E8 genome copies/ng
=1.5E10 genome copies/100 ng
This would be 10L of my stock, which is around 1E6 PFU/mL. What am I doing wrong here? Is my stock super dilute compared to an average stock? Did I miss something in my math?
Alternatively, what do you do to generate your standard curve?
Thanks!
= ~12kb ssRNA virus x 340 g/mole (average ribonucleotide MW)
I think you are missing some conversion from kb to grams to ensure units are cancelling properly.
Here's what I would do:
- Dilute series on viral stock
- Run plaque assay on dilution series
- Run RNA extraction on dilution series
- Perform RT-qPCR on each RNA extraction
You should now have two graphs with the concentration on the x-axis and either the plaque counts or the Cq on the y-axis.
Best of luck,
Erik
Hi Emma, i think the simple way for you to do absolute RNA quantification is to generate by yourself a synthetic RNA. It's quite simple and conveniant, I explain : You design a PCR system which flank your qPCR system, on the forward primer at the 5' you had the T7 promoter sequence (TAATACGACTCACTATAgggag). You run PCR, purification and then in vitro transcription ( https://www.thermofisher.com/order/catalog/product/AMB13345#/AMB13345). With you synthetic RNA you just quantifiy it with a nanodrop. As you know exactly the size of the amplicon and the quantity, you can generate a standard curve by serial dillution for all your experiment and perform absolute quantification. We do it for all our RNA viruses for performing growth curve kinetics, antiviral assay etc... and it works perfeclty fine. Good Luck and if you have more questions you can contact me.
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