how to get rid of mouse RNA - (Nov/11/2007 )

Hi guys,
i am going to do several microarray experiments on a pathogenic yeast after internalised by mouse macrophage cells. In order to extract total RNA of the yeast, I need to lyse the macrophages, so the internalised yeast can be released into medium. i had my first extraction, however, most of the RNA extracted was from macrophage rather than the yeast. Does any of you have managed to get high quantity of total RNA of any intracellular pathogen in the past? Is there anything I can do to get rid of the macrophage RNA? Any comments are welcome!!! Thank you in advance!

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I haven't done it, but have a idea. Yeast could not be lysed by AquaRNA without being first treated with lyticase/proteinase. So maybe you could try this:

(1) add AquaRNA (a potent lysing agent for mammalian cells) to lyse the macrophages as if you are extracting macrophage RNA;

(2) spin to pellet the yeast cells (may need to add a vol of PBS to dilute the lysate and help pelleting the yeast);

(3) wash the yeast pellet with serum containing culture medium to help degrade residual contaminating macrophage RNA (may need to incubate at 37*C for 10-30min between washes to aid RNase digestion);

(4) wash the yeast cells a couple times with PBS to enrich the clean yeast cells;

(5) now treat the purified yeast cells with lyticase/proteinase to break down the yeast cell walls and then go ahead using AquaRNA protocol to extract the yeast RNA.

This strategy might work for bacteria infected cells as well.

Good luck!

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