[mercier]

Ribominus

Hi wiggy

I have tried Ribominus and it does not work.. might be my RNA is degraded.. I have never analyzed 18S and 28S rRNA by qRTPCR. Could you please let me know how to do it?

Thanks
walle

Hello,

I'm trying to improve the rRNA depletion with the Ribominus for RNA-seq but at this time I can recover only 250 ng of rRNA depleted RNA with 10 µg at the beginning...
They say that normally I have to recover between 1 and 2 µg...
But the depletion works... Can I know how much you recover?

[neveaire]

Using the ribominus kit from Invitrogen, I generally recover between 5-10% of mRNA from total RNA (from human cell lines). I've been starting with 2-3 ug, so 250ng seems very low given your starting input. I have noticed that instead of incubating at 37C on a heat block, a water bath works much better.

[mercier]

Thank's for your reply... I've used first a heat block for the 70°C incubation and after directly transfert to a heat block at 37°C... Did you let the tube in the same bath and cold in it during the following 30 minutes?

[ETHANol]

There are a few threads here where people say RiboZero works way better then Ribominus.

[protist]

Thank's for your reply... I've used first a heat block for the 70°C incubation and after directly transfert to a heat block at 37°C... Did you let the tube in the same bath and cold in it during the following 30 minutes?

I have used and RiboMinus both for eukaryotic and bacterial libraries and have never achieved anything near the quoted depletion. I have been using RiboZero for the few months and have seen a vast improvement. For one set of libraries where the best depletion we could get with RiboMinus was a mere 20% (80% rRNA in seq reads) the same RNA treated with RibZero only had 5% rRNA contaminating reads. I tried many tweaks to get better results with the RiboMinus but was unsuccessful. For our bacterial libraries the RiboZero is more expensive than RiboMinus but the mRNA read number bump we get makes it worth it.

[neveaire]

Thank's for your reply... I've used first a heat block for the 70°C incubation and after directly transfert to a heat block at 37°C... Did you let the tube in the same bath and cold in it during the following 30 minutes?

So I used a heat block for the 70C incubation and did all 37C incubations (30 or 15min) in a water bath. It worked well for me using the eukaryotic kit, but others have had better luck with the Epibio ribozero kit. I've been meaning to try that as well, but if you get something that works, best to stick with it. Good luck.

[ramsemm]

Hi All,

I am interested in looking at the metatranscriptome of some soil samples. We are not sure if we will specifically target only bacteria or just look at mRNA from everything in the samples. But, I wanted to post here and see what more experience transcriptomics individuals think.

Originally, I looked into what to do if we were wanting to look at mRNA from everything in the soil, and it seemed as though Epicentre's exonuclease was the best bet. However, then I read a paper describing the biases associated with using exonuclease to remove rRNA.

So, then I thought we should just select one group either bacteria or eukaryotes, most likely bacteria. I am planning to use the MoBio PowerSoil RNA Isolation kit for the total RNA extraction. I have seen only two kit options for enrichment of bacterial mRNA, one is the MicrobeExpress kit and the other is the RiboZero Meta Bacteria kit. Now, I noticed with the MicrobeExpress kit they recommend you also use the MegaClear kit to remove 5S rRNA and tRNAs. Is this what others typically do? Also, is there any mRNA enrichment kit that also includes or pairs well with a DNase treatment? Or is this usually necessary?

My overall goal with these samples is to get total RNA extracted and enrich for mRNA. These samples will then be sent for sequencing on an Illumina HiSeq for a 2x100bp run. Does anyone out there have any suggestions on handling/processing of the total RNA to enrich for either overall mRNA (prokaryotic and eukaryotic; without significant bias) or just bacterial mRNA? I have been looking through the literature but there seems to be a lot of variability.

Thank you all in advance!