qPCR inhibition by ...???

[sofievib]

Hi all,

I've been doing some qPCR experiments lately and I am starting to have some problems. I hope somebody can help me.

The problem is that I don't have any amplification at all for a certain sample. The signal is just 'undetermined'. The primers I use are optimized and work for all my other samples. I also included housekeeping genes and also here I have no amplification.
I did a PCR (using titanium Taq) on the same samples for some of the housekeeping genes that were also included in the q-PCR and after putting the product on gel I got nice bands...
I also tested if the cDNA reaction went well. This was confirmed.
The only thing I can think of is that something in the sample is inhibiting the q-PCR reaction. I'll tell you something more about the sample.

First I isolated RNA by the use of Trizol (based on Phenol and Guanidine isothiocyanaat, a method from Chomczynski and Sacchi) from sciatic nerve from mice. I tested the RNA integrity (it was fine, not degraded) and I had good 260/280 and 260/230 ratios. After RNA isolation I performed a DNAse treatment to get rid of (possible) genomic DNA. I used the TURBO DNA-free kit from AB. Then I did a cDNA reaction and I used the Superscript III first-strand synthesis system for RT-PCR from Invitrogen.
I think the problem is the tissue itself. Does anyone know if nerve tissue contains something that could inhibit qPCR. It could also be something else, if anyone has some clues or hints...
Thanks a lot!!!