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Isolating bacterial RNA and protein from the host tissue sample


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#1 jcho130

jcho130

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Posted 23 November 2010 - 02:12 PM

[font="Verdana"][font="Verdana"]Hi,

Currently I’m carrying out a project which invovles isolation of bacterial RNA and its secreted protein from the infected host tissue.

As this is an in vivo experiment, I’m having difficulty in “efficiently” isolating the bacterial RNA and protein. My current protocol invovles

1. Suspend the dissected tissue in PBS and lyse using mechanical blade.
2. Pelleting of the cells in the lysate via centrifugation
3. Follow instruction provided from the used kit to isolate RNA and its protein.

I was wondering if there was a way for me to seperate bacterial cell from the tissue sample to bring up the efficieny of the isolation process.
Currently I get around 1 x 10^6 bacterial cells (from the whole lysate). When I centrifuge the lysate, I get ~1.5 cm pellet. Obvioulsy the pellet will be made up mostly of host tissue and its debris.

In a recent paper published, the author suspended the infected tissue and pipetetd up and down to detach the bacteria. Thus, ideally the pellet of the supsension will consist mainly of the bacterial cells. Would you suggest me to do the same?

Also is there any particular "kit" that you would recommend? A lot of people seem to have most success using Trizol.

Thank you for your time!





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