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Old 02-07-2014, 03:16 AM   #1
meggie.gr
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Default No 28S peak

Hey,

I am using Direct-zol™ RNA MiniPrep to isolate my total RNA from cells in TRIzol. I did isolate it and run bioanalyzer - results in attachment. I am clearly missing 28S peak, material is coming from M.lignano, flatworm. Any ideas? Bioanalyzer also gives an error: unexpected signal at 5S. Is my RNA degraded? Thanks a lot!
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Old 02-07-2014, 06:42 AM   #2
kwaraska
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While I know nothing about your organism, I do know that if you denature drosophila RNA the peaks are virtually on top of each other-more like a doublet than two distinct peaks-whereas if you don't denature it they resolve better.

Maybe try running it non-denatured and see what you get?
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Old 03-07-2014, 02:05 PM   #3
MLog
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"material is coming from M.lignano" - Do you mean Macrostomum lignano, a flatworm? I recently extracted RNA from another flatworm, Opistorchis, and it looks exactly the same.
THis is actually not rare for invertebrates, i've seen the same for snails, amphipods etc.
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Old 03-07-2014, 11:54 PM   #4
meggie.gr
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Quote:
Originally Posted by MLog View Post
"material is coming from M.lignano" - Do you mean Macrostomum lignano, a flatworm? I recently extracted RNA from another flatworm, Opistorchis, and it looks exactly the same.
THis is actually not rare for invertebrates, i've seen the same for snails, amphipods etc.
Yes, I am working with flatworms. I indeed see that more often while testing other flatworm species. In denaturing conditions the bigger subunit dissociates and forms two smaller, which seems to overlap with 18S. Thanks for the feedback!
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Old 03-07-2014, 11:55 PM   #5
meggie.gr
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Quote:
Originally Posted by kwaraska View Post
While I know nothing about your organism, I do know that if you denature drosophila RNA the peaks are virtually on top of each other-more like a doublet than two distinct peaks-whereas if you don't denature it they resolve better.

Maybe try running it non-denatured and see what you get?
Thanks a lot! You are totally correct. We confirmed that with running both: my total RNA and Drospohila RNA on the gel. Peaks looks similar too. Thanks again!
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