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Old 08-26-2010, 02:13 PM   #1
epibio
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Default Ribosomal RNA removal for Gram-negative bacterial RNA

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.
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Old 08-30-2010, 03:37 PM   #2
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Has anyone used Solexa to characterize bacterial transcriptomes from infected mammalian tissue?
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Old 09-01-2010, 05:57 AM   #3
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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.
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Old 09-13-2010, 06:13 AM   #4
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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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Old 09-13-2010, 07:49 AM   #5
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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.
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Old 11-28-2010, 05:00 AM   #6
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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?
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Old 11-29-2010, 08:44 AM   #7
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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.
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Old 11-30-2010, 12:28 AM   #8
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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?
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Old 11-30-2010, 07:45 AM   #9
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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.
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Old 11-30-2010, 01:20 PM   #10
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Have either of the Ribo Zero kits been tested with Mycobaterial strains?
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Old 12-01-2010, 06:31 AM   #11
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No, they haven't. If you're interested in evaluating a kit, please contact our UK distributor, Cambio Ltd.
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Old 04-07-2011, 04:12 AM   #12
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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)?
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Old 04-07-2011, 05:28 AM   #13
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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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Old 04-07-2011, 05:32 AM   #14
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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?
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Old 04-07-2011, 05:35 AM   #15
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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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Old 04-07-2011, 06:02 AM   #16
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The results are a little difficult to interpret. Initially for ten E. coli samples we started with 10 ug. After a single cycle of ribo-minus, the 23S and 16S rRNA looked to have been removed (by Bioanalyser pico-RNA chip analysis).

By UV-spectrophotometry (Nanodrop) it appeared we had what we considered quite good yields: 4-7.5%. However the Nanodrop spectra were questionable: there was a large peak -- probably remnant guanidinium from the "concentration modules" used post-depletion and most of the signal at 260 nm appeared to be a shoulder of that contaminant peak. Upon flurometric determination of the RNA concentration with Ribogreen we determined that our yields were actually 0.4%-1.2%.

Indeed this was less than the 200-500 ng recommended by the SOLiD total RNA seq kit when using ribo-depleted samples. Maddening.

I should add that we previously were seeing even lower yields from Ribo-minus. But I attribute this to substituting a heat block for a water bath in one of the steps. When we added water to the heat block well and allowed it to warm prior to the incubation (hence emulating a water bath) yields increased. Anecdotal, at best, since it had been months since our previous attempt with ribominus and that was with a eukaryote--but I can imagine there being a big difference between a water bath and a heat block.

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Last edited by pmiguel; 04-07-2011 at 06:05 AM.
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Old 04-07-2011, 06:29 AM   #17
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So the water bath seems to be better? I need mRNA for subtraction studies,no sequencing. I need about 2 ug and I wonder how much can I obtain from one reaction with 5 ug...
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Old 04-07-2011, 08:31 AM   #18
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In this instance.

Generally if a protocol calls for a water bath, a water bath should be used, if possible. But some protocols will be robust to heat delivery method. (Probably those with longer incubation times.) If you think about the nature of the energy delivery to a sample via a water bath and a heating block you could identify a number of differences. Especially if the heating block is not form-fitting to the vessel it holds.

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Old 04-08-2011, 08:15 AM   #19
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@mimila: Keep in mind the amount of mRNA in total RNA for bacteria is 2-5%, depending on the strain. For the Ribo-Zero Kits with 5 ug of E. coli total RNA, we typically see recovery of 350-600 ng. A "mock-treated" reaction gives about 4 ug after the procedure. The yields are highly dependent on the method used to purify the rRNA-depleted RNA; we recover about twice as much with Zymo columns compared to Qiagen.

@pmiguel: Although the Ribo-Zero method is a hybridization-capture method, it differs from Ribominus in that it is specifically designed to give good recovery with partially degraded RNA samples. For example, see this blog post.
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Old 04-08-2011, 10:15 AM   #20
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Yes, and I presume this design involves increasing the capture oligo coverage. More oligos probably. I suppose they might be better placed, somehow. But my guess is that instead of using X oligos to capture the ribosomal RNA, 2X are being used. (Or it could be 10X, I suppose.)

It would be useful to know the extent of degradation of the FFPE samples referenced in the linked site. Also would have been good to see the performance of "company A" on the FFPE samples.

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