troubles with efficiency in Taq-Man - (Jan/05/2007 )

Hi everybody,
I'm new here and I'm working for the first time with real time PCR, so I apologize for any stupid question, but I really need some help.
I'm using taq-man system, and I use probes form Roche. According to their website they should be optimal for my genes of interest. The primers are the ones designed by Roche, manufactured by a different company.
The RNA I used to make c-DNA comes from human post mortem samples, and to make my standard curves I used a DNA I made from commercial human reference total RNA.
I was checking 4 housekeeping genes, and 4 other genes I am interested in. The problem is that the efficency value that shows up next to my standard curve is low, around 1.4-1.5. I used the protocol suggested by Roche for the Light cycler, and I also tried (by mistake )a reaction mix with more primers.
Is there any way I can improve it? Can I trust results with low efficiency? Can the low efficiency depend on poor quality RNA?

I appreciate any sugestion


micetta

you absolutely cannot trust your results with efficiencies like that

have you set up the titration scheme for template and primers? there are some great tips in user bulletin#2, ABI. it's a long, dry read but there are some useful nuggets to be found inside.


and you are using a kit from one source with primer/probe set from another source? because of this, I would not assume that it's been optimized properly. I would assume in this situation that you will have to do some tinkering to get good efficincies

good luck