RT-qPCR of bacteria mRNA from sludge - RT-PCR, Real-time PCR, mRNA, sludge (May/10/2006 )

Dear all,

I've struggling this for long, but it seems that the inhibitors in the sludge is very strong and I only have very low abundance of the bacteria in the sludge.

What will you suggest to increase the PCR efficiency of a qPCR of low abundant genes in a mixed culture like sludge? (Do you have any idea which reagent is good at this?)

For RT, I wonder what reverse transcriptase is good. I've got clear bands for RNA isolated from sludge using TRIzol, but I was not successful in getting the cDNA using QuantiTect.

Any suggestion and help are desperately needed. I've spent months in figuring these out!

thanks,
Hang

-Hang-

QUOTE (Hang @ May 10 2006, 04:22 PM)

Dear Hang,
There are several question that you need to consider...
1. Have you tried to culture the bacteria from sludge? If you can you will get a graet abundance of bacteria that you dont have to worry about ther number and the inhibitor.
2. RT enzyme, MMLV is as good as any other.
3. How are you so sure that the band you see is from bacteria RNA? It cund be totally something else.... or from some other organism... this might be the reason you are not getting the cDNA using QuantiTect.

best regards

-Hadrian-

Why are you trying to do an RT PCR reaction on RNA from sludge? This seems to be about the most unlikely place to find intact RNA that I can think of. I'd like to know what your real goal is -- it sounds to me as if DNA extraction would be a much more likely path to find specific genes. As suggested above, isolation of growing bacteria would be another good approach.

-phage434-

Thanks a lot.

I am trying to quantify the active portion of a specific group of bacteria from the sludge in my reactor, that's why I need to do the RT-qPCR. As I need to monitor the changes of their quantification, it may not be possible to enrich them first.

I will check with the 16S rDNA first then.

Thanks....

-Hang-