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Old 10-26-2014, 05:34 PM   #1
Mike2188
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Default Weird RNA: Is this degraded or not?

I have been asked to prepare some sequencing libraries for a collaborator from pellets of psuedomonas bacteria that were in stationary phase. I isolated RNA and ran it on the agilent 2100 Bioanalyzer with an RNA nano chip. My results were fairly interesting, as there appears to be very little rRNA detected, however there doesn't appear to be any of the usual signs on degradation. For instance, the ribosomal peaks that do appear are very sharp, and there really isn't really much of a slope. I can think of four possibilities, do any of these make sense?

1) As the bacteria were growing in stationary, the rRNA was greatly downregulated as growth slowed.

2) Somehow the RNA extraction enriched for small RNA populations

3) Some small transcript is swamping out the rest of the ribosomal populations

4) The RNA is degraded

What do you guys think?
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File Type: png electropherogram1.png (48.8 KB, 36 views)
File Type: png electropherogram2.png (38.9 KB, 25 views)
File Type: png Electropherogram3.png (49.4 KB, 19 views)
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Old 10-26-2014, 06:48 PM   #2
nucacidhunter
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Could you post that run's full report (including other samples and ladder).
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Old 10-26-2014, 09:38 PM   #3
Mike2188
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Thanks for your interest,

Here are the full reports. You'll find the ladder and everything else looks normal. Some samples were run multiple times, over both chips, and look the same. Some samples were run at different dilutions, and still no change.
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Old 10-27-2014, 02:09 AM   #4
airun78
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Hi,

that looks degraded to me. The 23S peak should be higher than the 16S but in your samples it is the other way around. The 23S rRNA is more susceptible to degradation, that's why the ratio between 23S and 16S tells you if the RNA is degraded. The huge peak of small RNAs probably contains lots of degraded rRNA.
I would look into the protocol to see what can be improved: pellet storage and RNA extraction protocol.

Good luck!
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Old 10-27-2014, 06:20 AM   #5
Mike2188
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Thanks,

I have worked with low RNA quality samples before, with RINs ranging from 1-9 on the bioanalyzer, and I have never seen just one small and sharp peak like this before. I would expect RNA this degraded to be more of a smear, especially if some of the ribosomal subunit is still visible, or in some cases, where the small and large subunits are still in a 1:1 ratio (i.e. pme2 and pme2a on the first BA trace). The only difference is that I have never worked with degraded bacterial RNA, so maybe it is degraded? I will try to rework the protocol and I will post what I find out. If anyone else has any suggestions or ideas please let me know.
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Old 10-27-2014, 06:26 AM   #6
Mike2188
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In regards to my above comment, here is what electropherograms will typically look like in previous instances where I have had severely degraded RNA. However, these samples were heat and chemically degraded. Perhaps these bacterial samples were enzymatically degraded, which may explain some of the differences?
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Old 10-27-2014, 08:30 AM   #7
nucacidhunter
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I wonder if you have used Qiagen columns for extraction or purification of the samples.
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Old 10-27-2014, 08:41 AM   #8
Mike2188
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Nope, these samples are simple extracted using Trizol. Old school. I am going to try a kit to see if it changes the results later this week.
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