standards for copy number - please help (Nov/19/2006 )
I have been having problems making my real time PCR standards. I want to get copy number of my transgene per number of copies of GAPDH. This is what I have done so far.....
- extracted total RNA from cells that are transfected with my transgene
- reverse transcribe
- run regular PCR with transgene primers and and GAPDH primers
- purify PCR product with Quiagen kit
- measure dna on spec (260) for transgene and GAPDH
- use whole transgene sequence and primer sequences on BLAST to get length of amplicon
- do this for GAPDH too.
- work out number of molecules per ul using equation (A260/13.2 x s) x NA/10power12
- dilute transgene and GAPDH so that I have 10power8, 10power9 etc down to 10 copies per 5ul.
my reaction mix is usually 12.5ul SYBER green master mix, 0.1ul 5' primer, 0.1ul 3' primer, 7.3ul water and 5ul sample. Total of 25ul/well.
so I made my standards so that they would have the correct number of copies/5ul and then add 12.5ul SYBER green MM and 7.5ul water, no primers.
Is this the right way to make them? so the machine should jsut be measuring the dna already there as it binds to the SYBER green, no amplification?
So then when I put then on for a run on the 7900 I tell the machine they are standards and how many copies they are. However the machine doesnt make a curve, or doesnt seem to recognise them.
I really dont know what I am doing as I am totally new to molecular biology never mind real time PCR! And the lab that has the fancy 7900HT machine are not much help as they run most their samples using Ct.
Any help would be so much appreciated.
I think you maybe better off cloning your product directly into a topo TA vector and using that as the standard. Typically with RNA quantitation, I use an in vitro transcribed RNA as a template for the reaction. Therefore I am measuring both the RT and PCR reaction.
You need to include the primers in your standards as well. That's why the standard curve is not showing up on your real-time PCR run. Your transgene is being amplified from the sample and so it must be amplified from the standards as well. Then you can use the curve to determine the copy number in your sample.
I feel a wee bit stupid!
I thought that the primers were stuck to the the amplicon in the standards, and when you run the real time pcr reaction you dont need to add them again because they are already attached. Because you measure how much DNA you have by calculating the length of the amplicon including the primers in BLAST. I am a bit confused!
I have never heard of that method but it sounds good. Do you have some more info on how to do that?
Thankyou both very much for getting back to me