Dear all,
We’re currently extracting total RNA from gammaridean amphipods (Gammarus pulex). Animals were freshly collected, directly frozen at -80°C.
Trying different RNA extraction protocols (Qiagen, Trizol+Qiagen) we always end up seeing no obvious 28S band. First we thought it is due to the denaturing step prior to loading onto the Fragment Analyzer (known issue). But omitting this heat denaturing step does not lead to a change - the picture remains (see attachment for examples). Any ideas? We also gave the samples to a different lab - extraction results the same. There seem to be sometimes two only marginally different peaks at the 18S band position and we suspect that something other than heat is leading to the dissociation of the two 28S subunits possible. If these are of same lengths as the shorter 18S fragment can this explain the pattern… So non-model organisms experts: Can someone help us with this puzzle? Best Florian and Team
Hi Florian,
I know this phenomenon also from my work with protozoans. The LSU rRNA dissociates into 2 (sometimes ~equally sized) fragments due to a "labile" or "hidden break" region that undergoes a fairly rapid processing in vivo under denaturing conditions. But in my experience this also happens during non-denaturing gel-electropheresis or Bioanalyzer runs and might have already happen during the extraction. This "hidden break" is also known from insects, trematodes and fish. Maybe this causes your missing 28S band as well. A very gentle RNA extraction may could help. Ogino et al. 1990 (J Mol Evol, 30:509-513) or (Article Insects' RNA Profiling Reveals Absence of “Hidden Break” in ...
) might be interesting to read.
Best,
Sascha
Hi, sorry, I don't know how to explain this. But we are also puzzled by this phenomenon when we extract RNA from Brachionus calyciflorus Pallas. We compared different numbers of individuals, protocols and others. It seemed we always get only one band or the other is pretty faint. I'm wondering whether this is common for those small invertebrates? Anyway, I'm also looking forward to more reasonable explanation.
Hi Florian,
This seems very similar to problems seen in isolating RNA from pancreas. Is it possible that your organism has a high level of RNAase and the issue is degredation? Standard methods are insufficient to protect RNA in pancrease and you may have the same problem. There are several on line resources with suggestions on isolation of RNA from pancreas which might be useful. The attached image shows RNA isolated by standard methods similar to those you are using from 1) pancreas, 2) liver and 3)adipose tissue and is from a post on research gate last year.
https://www.researchgate.net/post/RNA_extraction_from_pancreas
I hope this is helpful. Good luck!
Frank
Hi Florian,
I was thinking maybe this is due to the less copies of 28S than 18S, which leads to higher concentration of 18S than 28S. Do you have the whole genome of your species? Maybe you can check the gene components. Or as Frank said there are too many specific RNAease which degrade 28S into small molecules.
Best wishes,
Yuzhan Yang
Hi Florian,
I know this phenomenon also from my work with protozoans. The LSU rRNA dissociates into 2 (sometimes ~equally sized) fragments due to a "labile" or "hidden break" region that undergoes a fairly rapid processing in vivo under denaturing conditions. But in my experience this also happens during non-denaturing gel-electropheresis or Bioanalyzer runs and might have already happen during the extraction. This "hidden break" is also known from insects, trematodes and fish. Maybe this causes your missing 28S band as well. A very gentle RNA extraction may could help. Ogino et al. 1990 (J Mol Evol, 30:509-513) or (Article Insects' RNA Profiling Reveals Absence of “Hidden Break” in ...
) might be interesting to read.
Best,
Sascha
Hi ! I am so interested in this problem as far as I am interested in cuticular degradation into onthogenesis in the culture insects for laboratory (microbiology, genetics - see Galleria mellonella). Could be an unknown phenomenon of infestation with an RNA virus. Sorry for my "uninformed" obs :)
We just published some observations of this phenomenon across arthropods:
https://www.researchgate.net/publication/330113327_RNA_profile_diversity_across_arthropoda_guidelines_methodological_artifacts_and_expected_outcomes
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