genomic DNA for qPCR quantification - target sequence? - (Jul/13/2007 )
I read a couple of posts, some of which are about variation in housekeeping gene (HKG) expression. It has been shown that levels of HKG (ActB, ActG, GapDH etc.) in a dormant/activated stem cell system in vivo are very different per single cell (around 100fold, PMID 16314490). Is it therefore possible to use genomic DNA as internal control? I'd like to use very small numbers (1-5 cells per reaction) of FACS-sorted cells, and I'm thinking of using something like satellite DNA (or any DNA sequence with a sufficient number of repeats per cell, so the signal is strong enough). With this, one could count the number of sorted cells and had an absolute measurement of mRNA molecules per single cell (one-step multiplex single-cell qPCR-combined PCR, it works sometimes ).
What do you think of this approach? Does anyone know some good target sequence? Should be open chromatin, repeat in sufficient high number, best would be fixed number inter-individually (not like paternity testing sequences, but they might also be ok, if one uses cells from the same mouse), what else? Has anyone heard of a paper dealing with this? Some other thoughts?
Any help will be highly appreciated!
Tough idea, but may work. I would worry a little about the effeciency of the RT reaction, as you are not accounting for this + it might be less efficient if you cannot clean your RNA from DNA. But hey, that would make a nice technical note.