Taqman for low levels of a gene (LacZ gene)in tissue - (Dec/02/2005 )
I have been doing Taqman to quantitate the amount of lacZ gene expression from muscle tissue. (I inject the muscle with AAV virus which has lacZ gene).
Some muscles have high copies & some muscles have very low copies of the lacZ gene. (This I know from muscle sections stained for betagal).
The problem I have is that the Taqman which is supposed to be very sensitive is unable to pick up the gene copies from some low expressed muscles.
Basically I extract DNA , just like we extract genomic DNA and do the Taqman.
I dont knwo if Im missing something.
The primers & probes r from previously published papers. So I am assuming they are fine
Please can someone give me any suggestions as to how to do a taqman with DNA from low expressed genes in tissues.
Thanks a lottt in advance
I think the first thing to do is to establish what your sensitivity is. You should use a standard cirve with a known copy number of target to do this. Since you are likely below single copy per cell for your expressed gene, you need to take into consideration how many total cells you are analyzing in your pcr reaction.You maybe only searching for 5-10 total copies.