Harvest intracellular bacterial RNA - (Jun/25/2009 )

Has anyone here ever done qPCR on intracellular bacteria to measure expression of genes? I'm looking to measure expression of a few bacterial genes involved in virulence, and I want to try to measure at different time points after infection of macrophages. I'd like to be able to measure early on when there won't be a lot of bacteria in the macrophages, but I'm worried the amount of eukaryotic RNA harvested would overwhelm my capability to measure the bacterial RNA. Later on in the infection process when there are more bacteria present, there shouldn't be a problem (I hope). Any thoughts on whether or not the eukaryotic RNA will or will not pose a problem, and if so any ways to either concentrate the bacterial RNA or eliminate the eukaryotic RNA?


I haven't done any of the work yet, but plan on using the RNeasy kit to harvest the RNA.


Thanks

Just to stimulate some thought... I'm thinking of way to approach this from a "ways to lyse the macrophages without lysing the bacteria" perspective. If you could get the bacteria out of the macrophages, you could do FACS or one of a multitude of pull-down assays. The problem I'm running into is that since this needs to be quantitative if you use a method that lyses both then you need to know what your yield of bacteria is (relative to how many were initially present), and that would be very difficult.

Reorganizing my thoughts towards a more PCR-centric standpoint, the answer to this question should be based on how good your primers are. Have you blasted your desired primer sequences to see if it may be an issue?

Cheers,
-Carlton

Carlton H on Jun 25 2009, 10:54 PM said:

We currently are able to lyse the macrophage without lysing the bacteria. Triton X-100 (1%) will lyse the macrophage to release the bacteria. As for FACS or pull down assays we don't have a multitude of antibodies for this pathogen yet. We have a few against LPS and other outer membrane things, but nothing specific for the proteins we're looking for in the macrophage (of bacterial origin). Furthermore, we expect these genes to be induced intracellularly, so I'm not sure if we'd get what we're looking for if we release them from the macs (would depend on how long afterwards we're able to sample). We've gotten expression in certain conditions in vitro, but want to see of that correlates to expression in the mac.

As for taking into consideration of yield, we had planned on measuring a bacterial endogenous (16s) as well as a macrophage endogenous (18s, B-actin, etc) to account for different numbers of bacteria. We could also see about doing gentamicin exclusion either in the same wells or in parallel wells.

That is, if I'm understanding your points correctly.


As for the primers, they work well for bacterial RNA isolated from broth cultures. Obviously it will be more difficult when dealing with the macrophage RNA as well. I have not blasted the primer sequences against the eukaryotic sequences, but since our host DNA is not completely sequenced, it would not be an exhaustive search, but perhaps one that would be useful to avoid potential problems.