[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!

[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.

[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.

[ETHANol]

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

[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?

[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]

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?

[torbean]

I see. Ok. Thanks.
Do you screen for rRNA contamination anyhow? PCR or so? I saw no bands on RNA gels for rRNA, but around 40% reads were rRNA after sequencing run

[protist]

A little question to the topic: it is stated in the Dynabeads Kit that eluting and re-binding mRNA extract to the washed beads removes rRNA concentration. Is it so?

I am hesitating whether to put Invitrogen RiboMinus on top, my mRNA concentrations are usually quite limited...

We have consistently used Dynabeads for all our Eukaryotic libraries - 2 rounds of purification - <5% are non mRNA. Most of our libraries start with 1-2 ug total RNA and we have never had any problems. For Bacterial libraries I have used RiboMinus but have recently switched to RiboZero kits as they have seriously out performed the RiboMinus for some of our bacteria of interest. For your library prep I would recommend as few unnecessary manipulations to the RNA as possible and 2 rounds of polyA selection followed by RiboMnus seems a bit excessive to me...

[torbean]

A little question to the topic: it is stated in the Dynabeads Kit that eluting and re-binding mRNA extract to the washed beads removes rRNA concentration. Is it so?

I am hesitating whether to put Invitrogen RiboMinus on top, my mRNA concentrations are usually quite limited...

[irit]

I have used this kit a few times. For really effective removal of ribosomal RNA (as seen in bioanalyzer track) I had to do 2 rounds of ribodepletion. I had about 20% recovery (according to nanodrop, so take it with a grain of salt).

Do you use glycogen or some other carrier for the precipitation? The biggest problem is to miss your pellet because it isn't easily visible.

[neveaire]

ribodepletion using invitrogen ribominus

I'm going to attempt a ribodepletion using Invitrogen's RiboMinus Eukaryote Kit for RNA-Seq. Has anyone used this? Do you typically do the concentration step afterwards?

I tried it once with about 3ug input and noticed that without concentration the yield was simply too dilute. After concentration it was better, but the recovery was only about 2% of the input.

Thoughts appreciated.

[hoytpr]

I've been working with microarrays since they were invented. Now I am beginning RNA-Seq. Microarrays are not dead yet... obviously, because the problem of rRNA overwhelming your NGS data hasn't been solved. From reading, the Epicentre kit works well at removing rRNA, at least for human, mouse, and rat. But I am currently working on a transcriptome project with bats. It may be a long time before Epicentre gets around to a bat-kit. We will have to start with DNS-treatments, and obvious methods like rRNA subtractive hybridization. Nonetheless, I firmly believe that the future of transcriptional profiling is by HTS exclusively.

Microarrays done correctly (CORRECTLY!) give excellent results (albeit not quantitative). But anyone printing their own microarrays better know what they are doing and do a lot of testing. Microarray printing will persist in the world of protein-protein interaction screening (and protein-DNA screening too). But only as a screening tool, validated later by SPR.

Sorry about my first post sounding like a rant.

Peter Hoyt

[li zhang]

Li Zhang: have you considere using a microarray? Depending on what you want to do the cost, time to prepare samples and time for bioinformatics could all be significantly cheaper on an array.

RNA-Seq will give you more information but right now I would say you have to work pretty hard to get it.

Comments anyone? Microarray vs sequencing; are microarrays dead?

I wanna examine transcriptome including novel transcripts, I am told microarray is not so sensitive

[HESmith]

I don't think microarrays are dead, but their prognosis is poor in the long term. Sequencing offers some serious advantages in terms of sensitivity, flexibility (not limited by chip availability), and cost (which will only continue to decrease). The big disadvantages are the sheer amount of data (25K genes vs. 25M+ reads) and relatively steep learning curve for mastering the analysis tools. But, much like microarrays, off-the-shelf software suitable for bench scientists (no disparagement intended; I consider myself one) will make the transition easier.