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Isolating bacterial RNA and protein from the host tissue sample


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#1 jcho130

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Posted 23 November 2010 - 02:13 PM

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!

#2 bob1

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Posted 24 November 2010 - 02:33 PM

How about a centrifugation gradient to get rid of the majority of the host DNA and leave the bacteria as a discrete band?

#3 jcho130

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Posted 24 November 2010 - 06:20 PM

I did my homework for a couple of days and there seem to be couple of methods that I can undertake.

1. Differential centrifugation (using sucrose, ficoll-paque etc)
2. Differential lysis (treat the pellet with triton-X or saponin or just normal lysis buffer)
-Centrifuge then wash twice with PBS

I just tried out differential centrifugation with ficoll-paque and I managed to only salvage 1.6/100 of bacterial cells that I started out with. I tried out with bacteria only, bacteria + monocyte, monocyte only. I know by searching literature that bacteria I work with is trapped in the same layer as the monocytes. One other thing I noticed was that bacteria layer is "transparent" = I can't see a thing!!
I will try it out with tissue lysate next week and see how it goes. Obviously there are a lot of factors that I would need to consider...

I'm still open for suggestions :D

Edited by jcho130, 24 November 2010 - 06:30 PM.


#4 biocrazy

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Posted 26 November 2010 - 03:02 AM

I did my homework for a couple of days and there seem to be couple of methods that I can undertake.

1. Differential centrifugation (using sucrose, ficoll-paque etc)
2. Differential lysis (treat the pellet with triton-X or saponin or just normal lysis buffer)
-Centrifuge then wash twice with PBS

I just tried out differential centrifugation with ficoll-paque and I managed to only salvage 1.6/100 of bacterial cells that I started out with. I tried out with bacteria only, bacteria + monocyte, monocyte only. I know by searching literature that bacteria I work with is trapped in the same layer as the monocytes. One other thing I noticed was that bacteria layer is "transparent" = I can't see a thing!!
I will try it out with tissue lysate next week and see how it goes. Obviously there are a lot of factors that I would need to consider...

I'm still open for suggestions :D


Why dont you check the sedimentation coefficient of your host cell, debris, bacteria. I am telling out of my textual knowledge (not from my experimental experience). Just I wanted to give a clue. If you think this can help you then try it.




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