How do I know copies of RNA template added in a reaction ? - (Aug/08/2007 )

Hi, all.

I would like to test sensitivity of a real-time PCR protocol.
But, I don't know how do I know copies number of viral RNA template added in a reaction.

Help me, please.

-Kidd-

check quantitative (real time) RT PCR protocols. I'm not too familiar with them, but i know that, in comparison with references, you ma be able to know how much copies of RNA you have in your tube.

-fred_33-

Thank you very much Fred33. But, I'm sorry. I'm not clear explaination.

I mean I don't know how to measure density of RNA (copies) in a template solution. So, I cant make a standard for use as reference or anything.

Can I measure RNA density as ng/ul, then calculate copy number by devided with molecular weight of a whole viral sequence from GenBank ?

-Kidd-

well this is possible theorically. Measure OD, and then convert it with Biomath Calculator or sthg similar.
You need 3OD measurements at least.
2 limits :
first the conversion between OD and concentration is valuable for single stranded RNA. I don't know the molar extinction coefficient for dsRNA
second, you need to calculate the error percentage. take in consideration a 0.01 error in OD reading (or 0.005 if you have good spectrophotometer ; check supplier for variations in OD, and you may check also calibration of wavelenght). Then you have to calcultate the copy number of RNAtemplate this value represents. You may see it's relative high number, and thus meaning your method is not valuable for your purpose.

-fred_33-

Thanks again, fred_33.

How about it if I would like to measure copy number of a plasmid ?

Can I use this method ?

-Kidd-