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-   -   Degraded RNA from bacteria biofilms following Ribo-Zero (http://seqanswers.com/forums/showthread.php?t=66394)

JakobHedegaard 02-19-2016 03:29 AM

Degraded RNA from bacteria biofilms following Ribo-Zero
 
Hi,
We have applied Ribo-Zero to two sets of RNA isolated from bacteria. Set A was isolated from bacteria grown as biofilm and set B was from bacteria in culture. The total RNA is fine from both sets (RIN 9-10) but following Ribo-Zero the RNA from set A (biofilm) is degraded while the RNA from set B looks fine. As all Ribo-Zero reactions was conducted in the same batch, I am confident that "something" is present in the RNA from biofilms which make the RNA degrade during Ribo-Zero treatment.
Appreciate suggestions to troubleshoot.
Cheers,
/Jakob

adam.geber 02-22-2016 10:09 AM

Hi Jakob,

What method are you using to extract your RNA and how are you storing it (i.e. buffer, conditions, etc.)? If your extraction is yielding high-purity, intact RNA and both sets are stored and processed under identical conditions then the issue is likely caused by environmental contamination by RNases. Can you post representative traces from your samples, both before and after Ribo-Zero treatment? Also, what Ribo-Zero kit are you currently using and can you comment on the type of culture (i.e. single-species vs. co-culture)?


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