absolute qPCR problem - (Feb/06/2008 )
I have a big problem i can't resolve alone.
We are working on DNA and we have to quantify DNA (amount
of a given fragment) by real time PCR.
The problem is that we can't use a housekeeping gene to normalize because in one assay we will have DNA of this housekeeping gene A (but not the second one named and in other assay we will have to choose another housekeeping gene B (because the first A isn't at the same "expression" level in the second assay, inversely for B in the first condition) so we can't have the same housekeeping gene fragment to amplify as a "normalizator".
After reading litterature i think we should use absolute quantification (with a standard curve for the first condition and a standard curve for the second condition).
My question is, can i use absolute quantification in ng/µL (and not in copy numbers) without normalizing at the end?
Thanks for people who know the answer
What about making an artificial internal control template out of lambda DNA.
Tambong, J.T., Mwange, K.N., Bergeron, M., Ding, T., Mandy, F., Reid, L.M., and Zhu, X. (2008). "Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan ® real-time PCR assay targeting the cpsD gene.", Journal of Applied Microbiology, 104(5), pp. 1525-1537.