Intracellular bacteria RNA extraction - (Apr/30/2008 )
I am currently trying to optimized RNA extraction from intracellular bacteria. I am using U937 and have tried lysing the cells with 0.1% SDS and Triton and a mixture of phenol and ethanol to stabilise the bacteria RNA. However, SDS reduces my bacteria viability and Triton cant lyse well after incubation for 30 min on ice. I am now trying to lyse with 4% saponin in 50% PBS and 50% RNAlater. However, I discover that much of the cells were not lyse well after 10 min at RT and I have eukaryote RNA carry-over. Does anyone has any experience in this? Can anyone help me? Thank you!
-syLoc-
QUOTE (syLoc @ Apr 30 2008, 12:37 AM)
I am currently trying to optimized RNA extraction from intracellular bacteria. I am using U937 and have tried lysing the cells with 0.1% SDS and Triton and a mixture of phenol and ethanol to stabilise the bacteria RNA. However, SDS reduces my bacteria viability and Triton cant lyse well after incubation for 30 min on ice. I am now trying to lyse with 4% saponin in 50% PBS and 50% RNAlater. However, I discover that much of the cells were not lyse well after 10 min at RT and I have eukaryote RNA carry-over. Does anyone has any experience in this? Can anyone help me? Thank you!
You should not use 50% RNAlater, it is like 3M salt and will "fix" your cells. You can't get your bacteria out this way. Try this:
1) Pellet your cells,
2) Lyse your cells in 0.025%SDS
3) Spin and rinse your bacterial pellet with 0.025%SDS 3x
4) Extract baterial RNA per your protocol (AquaRNA is the best!)
Good luck!
-chessplayer-