Ribosomal RNA removal for Gram-negative bacterial RNA

[epibio]

EPICENTRE recently added a new ribosomal RNA removal kit to its lineup. The Ribo-Zero™ rRNA Removal Kit (Gram-Negative Bacteria) removes >99% of the 23S and 16S, and >97% of the 5S rRNA from both intact and partially degraded RNA from Gram-negative species. A kit for Gram-positive species is in the works and should be available soon.

[kwalters]

Has anyone used Solexa to characterize bacterial transcriptomes from infected mammalian tissue?

[epibio]

Not with this kit, so far. Theoretically, you could use oligo (dT) to get rid of eukaryotic mRNA, and then remove ribosomal RNA with two passes (eukaryotic and bacterial). However, you'd be dealing with very small amounts of bacterial RNA.

A better solution may be Ambion's MicrobEnrich kit, followed by the RiboZero kit. With either approach, you could prepare Illumina-compatible libraries for sequencing.

[pmiguel]

What sorts of yields do you get from E. coli RNA?

We have been trying to use Invitrogen ribominus but our yields are abysmal. We start with 10 ug of RNA and usually end up with less than 100 ng (by fluorimetry) after a single pass. (For the SOLiD, two passes and 200-500 ng of ribo-minus RNA are recommended.)

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Phillip

[epibio]

From 1-5 micrograms E. coli total RNA, we get around 130-600 ng rRNA-depleted RNA, after one round of treatment, as measured by RiboGreen. Bioanalyzer measurements give slightly higher values.

[mimila]

Is it possible to get evaluation kit from epibio? I have bought couple of times the earlier kit (mRNA only) but it gave to small amounts of mRNA. Is the new kit more efficient?
Martha

[epibio]

The Ribo-Zero kits use a completely different method from the mRNA-Only kits for rRNA removal. They are more efficient in terms of rRNA removal. The yield will depend on your sample type and purity.

We do have an evaluation program in the U.S.; for other countries, you would need to contact your local distributor.

[mimila]

Thank you for an answer. I can not find how that kit works.
Is that method similar to the microbexpress? I used microbexpress and it did not work for me (the results were worse than with mRNA only)
I work with Gluconacetobacter (Acetobacteriaceae). Do you have any information for usage this kit in bacteria different than E.coli?
Is ribozero enzymatic or hybridization method?
Martha

[epibio]

The Ribo-Zero kits do not use enzymatic digestion, but are based on a proprietary hybridization technology for rRNA removal. The Gram-negative kit will work with a broad range of species, including Gluconacetobacter. I'm not sure why the MicrobExpress kit didn't work for you; it might be a good idea to troubleshoot that with Ambion tech support before trying a different method.

[protist]

Have either of the Ribo Zero kits been tested with Mycobaterial strains?

[epibio]

No, they haven't. If you're interested in evaluating a kit, please contact our UK distributor, Cambio Ltd.

[mimila]

What is the average quantity of mRNA obtained from 5ug of RNA from bacteria? Do you have any values? I know that depends on organism but I would like to know what can I expect. Is the quality of total RNA important (I mean integrity)?

[pmiguel]

The 5th post in the thread answers your question for E. coli.

I don't work for Epicentre, but Ribo-zero looks like it is in the same "family" of ribo-depletion methods as Invitrogen's Ribo-minus. If so, it would work by hybridization to ribosomal RNAs by oligos attached to magnetic beads. Subsequent removal of the beads then also removes the ribosomal RNA. Okay, that leaves out a step, but the basic idea is there.

We typically got very poor yields when using ribo-minus. I have no idea why. Maybe something in our RNA preps in concert with something in the kit was degrading the non-bound RNA? Or we were getting non-specific binding?

Anyway, ribo-depletion methods that rely on oligo hybridization will be less capable of removing rRNA that is degraded to the extent that coverage of the oligos on the rRNA is less than 100%. The fewer oligos that bind to your species's rRNA, the more critical that the rRNA be perfectly intact to allow its removal.

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Phillip

[mimila]

Thank you Phillip,could you tell me what do you mean writing poor? For example how much is it from 5 ug of total RNA? Less than 0,5 ug?

[pmiguel]

0.5 ug from 5 ug is a 10% yield. 10% is near the maximum I ever see for non-ribosomal RNA in a sample. I can check to see what we were getting for ribo-minus on E. coli.

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Phillip