Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Copy number calculations in real time PCR - (Apr/17/2009 )

Hey all

I have a stupid question regarding calculating copy number in real time PCR.
If I have 30 ng/ul in an RNA extract (measured by nanospec). I dilute it to 10-6. I take 4 ul of that dilution to my PCR reaction ( 50 ul reaction). How do I know how many RNA molecules per reaction I have there? (i.e. how many many copies per reaction my system is able to detect). My amplicon on 120 bp.


Thank you very much for your help!

Anders

-Jka83-

Hi

Well, if you really want to do absolute quantification and you only have the 30ng/g value, I guess you'll have to know the specific weight of one molecule. If you know the sequence you can calculate the specific weight and then devide your 30ng by this value. But this is not very exact as you first will have a bias in the concentration measurement and second you don't know if you had a 100% reaction efficiency during your reverse transcription.
Don't know what you are planning to do, but maybe there are better ways to circumvent this problem. Why not just comparing the samples relative to each other?

good luck

Jka83 on Apr 17 2009, 04:50 PM said:

Hey all

I have a stupid question regarding calculating copy number in real time PCR.
If I have 30 ng/ul in an RNA extract (measured by nanospec). I dilute it to 10-6. I take 4 ul of that dilution to my PCR reaction ( 50 ul reaction). How do I know how many RNA molecules per reaction I have there? (i.e. how many many copies per reaction my system is able to detect). My amplicon on 120 bp.


Thank you very much for your help!

Anders

-littleaxt-

you need some kind of standard of your target RNA eg. in vitro transcribed RNA which you can quantify and calculate the copy numbers. this RNA is then reverse transcribed like your samples. some people also spike an inert RNA into the IVT RNA like tRNA to have the same RNA concentration in every reverse transcription reactions.

-tea-test-