[rdesprat]

Question

What is the level of degradation of RNA induced by the kit?Many people are telling me that they are getting degraded rna at the end of the ribozero kit...do you have a way to check that?

[SeqMonster]

You can use Agilent Bioanalyzer RNA chip to check your RNA integrity.

[NextGenSeq]

Since the first step in making an RNA-Seq library is fragmenting the RNA I don't see why degradation would be a problem.

[epibio]

We haven't seen degradation when testing for common transcripts on Northerns. You do retain the small-RNA fraction after purification if you use the recommended Zymo columns; perhaps this is being confused for degradation products.

[SysBioNaMA]

Hello,

I am involve in mixed microbial community research and i plan to realise a deep sequencing mRNA on total bacterial popuation. You propose two different Ribo-Zero rRNA removal kit for bacteria, one specific for Gram positive bactria and an other one specific to Gram negative bacteria. Nevertheless I interessted by rRNA removal on total RNA present. Could you tell me which kit match best with my project?

Tanks

[epibio]

At this point, my recommendation would be one round of each kit. However, we are working on a kit that will be suitable for total bacteria populations. I'll get in touch with you if you're interested in our beta-testing program.

[joanne.selway]

I am currently performing some LCM on human samples and wish to deplete rRNA before embarking on amplification for RNAseq. Do you think this kit would be appropriate, is it compatible with different amplification steps such as NuGene?

Jo

[epibio]

We have a low-input version of the Ribo-Zero (H/M/R) kit; however, it still requires 100 ng total RNA. It would not be suitable for single-cell amounts of RNA. If your RNA amplification procedure is designed to amplify mRNA, having the rRNA present shouldn't be a problem.

[mnkyboy]

Although the current kit is not configured specifically for low input, we do have customers who have scaled the protocol for use with ~50 ng total RNA. You would need additional spin filters since you're processing more samples than the kit was designed for, but it will work.

Can you tell us what type exactly the spin filters are?

[epibio]

We don't release manufacturing information, but any generic 0.22-micron sterile microfilter units would work. They are merely designed to remove the microspheres from the reaction.

[madseq]

Would this kit work removing rRNA from yeast (Saccharomyces)?

[NextGenSeq]

Invitrogen has a yeast RiboMinus kit

[epibio]

Would this kit work removing rRNA from yeast (Saccharomyces)?

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.

[Rogue]

Hey there,

I work on a host-parasite interaction where the parasite (Gram-negative) is intracellular. I want to sequence its transcripts but I have no idea what the proportion should be from the total RNA one extracts from the host... I wanted thus to remove all host's rRNA and this kit looks like convenient. The host is Drosophila: do you think the kit would work?

Thanks in advance

[epibio]

The H/M/R kit will work reasonably well with Drosophila (see our blog post). If you wanted to remove the parasite's rRNA as well, you could try a combination of the H/M/R kit and the Gram-negative kit. It would require some optimization to get the right ratio of rRNA removal solutions for your samples.