[pmiguel]

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.

--
Phillip

[mimila]

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...

[pmiguel]

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.

--
Phillip

[epibio]

@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.

[pmiguel]

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.

--
Phillip

[epibio]

We can't provide more detail on the capture mechanism, unfortunately, since the method is proprietary. Also, we didn't try the FFPE samples with "company A" since their method requires high-quality RNA.

More detail on FFPE samples is in our poster recently presented at AACR (1.2 MB PDF).

[mimila]

Does anyone do polyAtailing after obtaining mRNA from bacteria? I have some questions:
1) what is the minimal quantity of mRNa for polyAtailing. The protocol epicentre is designed for 1-10ug (alternative protocol) but I wonder that lower concentration are also good, for example 0,3 ug?
2) Does anyone check the shift after polyAtailing on the 1% denaturing gel? I have some problems because I do not see the shift between mRNA and mRNA polyAtailed and I wonder if it may look like that (bacuase reagents of kit are good for sure and mRNa is also good-purified with minelute Qiagen)

I will be ver gratefull for answers. I would like to start cDNA synthesis with oligoDT but I am not sure about my last step with polyAtailing. If I do not see the shift, what to do?
I hew new isolations, new kit...
HELP me,please

[flashton]

Has anyone tried the RiboZero kit for bacterial RNA? What were the yields like?

I have tried Terminator Exonuclease (TEX) from EpiBio and found strong, non-specific RNase activity, contacted the company and they said that they no longer recommend TEX for rRNA reduction (although their website doesn't yet reflect this).

Thanks,

Phil

[protist]

I have just had a student try the Gram Negative RiboZero kit on one of our Legionella samples. Previously we have used another rRNA depletion kit with varied but by no means spectacular success. We ran an Agilent RNA chip on the RiboZero untreated versus treated RNA (the student treated 2.5 ug total RNA) and I was very pleasantly surprised - the treated had virtually no SSU/LSU rRNA peaks and a reduced putative 6S peak - in fact to my surprise it looked very much like the EpiBio publicity shot! The student is currently in the process of converting the material into a strand-specific RNAseq library.

[flashton]

Thanks!

Do you know how much RNA was recovered?

[protist]

~ 100 ng but that is based on the Agilent concentration estimate. As I was so pleasantly surprised with the apparent complete depletion I wanted to get a library made asap - so got the student to go straight to fragmentation will all of the rRNA depleted material

[greigite]

Interesting thread. I thought I'd share our practical experience with rRNA depletion from microbial RNA extracted from soils. We compared epicentre's previous enzymatic-based kit (mRNA only prokaryotic mRNA isolation kit) with the Ambion microb express kit and had much better results from the Ambion (no visible 16S/23S peaks on the Bioanalyzer). We would typically get ~10 ng mRNA output out of the Ambion kit with total RNA inputs > 100 ng.

[flashton]

very interesting, thanks protist.

i'm running 12 samples multiplexed on one SOLiD 4 lane, expecting 35 gigabases of data (take from this paper*), divided by 12 samples is nearly 3 gbp of sequence per sample. even if 90% of that matches rRNA that still leaves me with 300 mbp matching mRNA or nearly 100x coverage of my genome (i love bacteria). so, my provocative question is - is rRNA reduction really necessary? im playing it safe this time but would be interested to see a comparison of rRNA +/- samples.

*http://seqanswers.com/forums/showthread.php?t=11511 im guessing the values for 'per run' on a SOLiD includes both lanes.

[protist]

Ah but flashton imagine how many you could get in a lane if 90% matched mRNA...

To rRNA deplete or not to deplete is a question we have argued back and forth in the lab. For us as we were working with infection samples we had host contamination as well as bacterial rRNA sucking up reads and a GAII not a HiSeq so it made sense then..now I am little less enamored... However a rRNA depletion protocol that works effectively (eg similar to our polyA selected material) and reproducibly thus allowing me to put even more Beasties per lane will get my vote It as yet remains to be seen if this is the case with our test Legionella samples - hoping to get a run on next week so my opinion may change based on the results! That said I have ordered their Gram Positive kit for another project...

[msdl]

mRNA Low yield with an excelent depleting

Hello,

We are having problems in the obtention of pure mRNA yield from total RNA of a gram negative bacterium (Chromohalobacter salexigens).

We tested Microbexpress from Ambion and the depleting grade wasn't enough to sequence it. Then we tested Ribo-zero (epicentre) and we obtained a succesful depleting grade (99% more or less) but the problem is, taking into account that the initial RNA quantity is 5 ug, we got only around 50 or 70 ng of mRNA (using the suggested kit of Zymoresearch to concentrate it) and it isn't enough to be sequenced.

The question is... How tricky could be to use the partially depleted samples in Microbexpress (apart the low depleting grade, the initial total RNA is up to 10 ug) to be depleted by the second one (ribo-zero)?

Any other suggestion to improve the mRNA yield knowing that the total RNA RIN is at about 8-9...?

Thanks in advance.