[mimila]

I know that quantity of sample for the reaction is up to 5 ug but I wonder that little more makes a big difference? I need to get so much mRNA as it is possible so I think about setting reaction with 8 ug. What can I espect after that kind of modification? I would be grateful for an answer.

[epibio]

Using more than 5 ug would potentially overload the reaction, so you'd see reduced removal of rRNA. I'd recommend splitting your sample into two reactions, and then combining the final output.

[hklein]

RiboZero on D. melanogaster

Hi there,

I'd like to assess the effect of the application of RiboZero on D. melanogaster samples.

Does anyone know of public D. melanogaster RNA-Seq data sets, where RiboZero has been used in two rounds? I'm especially interested in the complexity of the resulting libraries. Can anyone share experience with this?

Cheers,
Holger

[somi06]

environmental sample

Hi, I am considering using the product for an environmental sample for total rRNA removal (Eukaryote, Bacteria and Archaea) for RNA-seq. What do you suggest I do? Repeat rRNA removal using different kits?

Cheers,

[SeqMonster]

I am not a big fan of repeating treatments since you will probably loss more RNA through the repeated steps. One round of the Ribo-Zero Meta kit is sufficient to remove most of the rRNA and my sample is microbial infected human-associated sample. Ambion kit did not work very well on my human-associated samples. I have only one sample that would never work on any rRNA removal kits, and I still don't know why.

[somi06]

Hey SeqMonster,
So in your samples, there would undoubtably be some Eukaryotic rRNA right? Did use of Ribo-zero meta-bacteria kit get rid of Eukaryotic rRNA as well?

My understanding of the way the kit works is that it would only get rid of bacterial rRNA (23S/16S/5S) with bacterial kit...so in the case of samples that can have eukaryotic contamination, i would have to do two rounds of Ribo-zero subtraction with different kits BUT I could be (and hope) wrong!

[Olaf Blue]

It is certainly possible to mix two different rRNA removal solutions from the Ribo-Zero Human/Rat/Mouse Kit and the Ribo-Zero bacteria rRNA removal kit for depletion of "mixed" samples, followed by using the blended solution in a standard procedure in the protocol. One will reduce the amount of total RNA in the procedure (use 2.5 µg vs 5 µg). The ratio of the mixing of the rRNA solutions is recommended to be 1:1, but a "custom" ratio of human to bacterial removal solutions can be done if you have an idea of the relative abundance of bacteria in the samples.

[SeqMonster]

Hi somi06,

The meta kit is designed to remove both eukaryotic as well as bacteria rRNA, that's all I used. The meta kit works well in removing the human DNA in my samples.

[Olaf Blue]

Hi SeqMonster:

If you are referring to the Ribo-Zero Meta-bacteria kit, it is only designed for both gram positive and gram negative bacterial rRNA. Any human removal you get with this product is purely serendipitous. A soon-to-be released Ribo-Zero kit ("Epidemiology") will have capture probes for gram negative and gram positive bacteria as well as human/mouse/rat rRNA.

[SeqMonster]

Ops Sorry. My bet on the naming. Yes, Olaf blue was right. I was referring to the "Epidemiology" Ribo-Zero kit, which I think everyone is looking forward to its release! It is an extremely useful kit for researchers doing shotgun metagenomics study!

[Irshad]

rRNA removal kit for Gram negative bacteria

Hi there,
I am working with bacterial fungal interactions. I am supposed to do RNA-Seq, to see differential gene expression patterns of bacteria. Is it necessary to remove rRNA before sequencing??
If yes then which kit is best suited for my sample, with little risk of RNA degradation??

[jzx2002]

rRNA removal for Mycobacterium tuberculosis

Please contact me using the link in my profile and I'd be happy to set you up with the evaluation program. Thank you.

Hi,
Do you know which kit is good to remove rRNA from MTB? We have problem by using RiboMinus kit (Cat# AM1905).
Thanks,

[Olaf Blue]

Hello: the information we have with Ribo-Zero abd Mycobacterium tuberculosis is this:

"“RZ Bacteria - (Mycobacterium tuberculosis) Efficiently removes 23S, 16S and 5S rRNAs.”

Olaf

[Ivy]

rRNA removal from yeast

The H/M/R kit will give you >99% rRNA reduction from yeast, as long as you have high-quality (intact) RNA. If you're interested in beta-testing a yeast-specific kit, please send me a PM or contact me using the link on my profile page.

Hi. I'm trying to use the Ribo-zero rRNA removal kit(H/M/R kit) for rRNA depletion from yeast RNA. After sequencing, the rRNA levels left in my samples ranging from 15%-48%. Do you know what's the problem? Thanks.

[Olaf Blue]

Hi Ivy,

The post you're is rather old, and as time has gone on we have found that (as many already say) that the Yeast rRNAs are simply too diverse to be able to be removed completely using the RZ H/M/R Kit. While some yeast species do work well with the H/M/R Kit, we have found that other less common strains are not as easily depleted.

There is a RZ Yeast kit under development, but you can also contact Epicentre's TS and submit your sequences for sequence checks against the capture probes in the kit.

By the way - which rRNA species are contributing the sequences in the 15-48% fraction?

Olaf