[arabidopsis]

I thought about Experion, but there was not one available here. However, RT PCR worked on depleted samples, meaning that some RNA was still there. At least it shows that rRNA has been effectively removed

[li zhang]

Hi, Im a beginner at sequencing. I have several might silly questions. Are RNA-seq and transcritome sequencing the same thing and using the same protocol? Can paired-end squencing be adopted in transcritome sequencing? If so what is the protocol? Much appreciate!

[TonyBrooks]

Hi, Im a beginner at sequencing. I have several might silly questions. Are RNA-seq and transcritome sequencing the same thing and using the same protocol? Can paired-end squencing be adopted in transcritome sequencing? If so what is the protocol? Much appreciate!

RNA-seq and transcriptome sequencing are essentially the same protocol, but with different aims.
With RNA-seq, the aim is to estimate transcript/exon, and hence expression, levels. For this, you typically require lots of reads in order to get sufficient sensitivity.
The aim of transcriptome sequencing is to sequence the transcriptome - i.e. you're looking for polymorphisms in sequence, depth is much less of an issue.
Of course, you can "do" transcriptome sequencing with RNA-seq data, but not necessarily vice versa.

With regards rRNA sequencing, we have always used PolyA+ selection. Our experience with ribominus is that it doesn't remove sufficiently enough RNA for sequencing.
We're also just about to sequence a DSN treated library. We've been told that this method has excellent results for rRNA removal. It'll be interesting to see the data.

[li zhang]

Thx, Tonybrooks. Actually we wanna detect which genes are active during a specific period and in a particular tissue, and identify these expressed genes which are responsible for a particular physiological phenomenon. Does transcriptome sequencing work?

Could anyone answer my question about the paired-end sequencing part?

[TonyBrooks]

Thx, Tonybrooks. Actually we wanna detect which genes are active during a specific period and in a particular tissue, and identify these expressed genes which are responsible for a particular physiological phenomenon. Does transcriptome sequencing work?

Could anyone answer my question about the paired-end sequencing part?

It looks like you need to do an RNA-seq experiment as you're looking for differential expression. You'll need a control sample to compare, same tissue type from a "healthy" individual. We've found transcriptome sequencing to be a very powerful tool. It's obvioulsy more sensitive than microarrays, (background is essentially zero), gives potential for identification of novel transcripts and alternative splicing events (that may not be targeted on an array), plus you have the added bonus of identifying any polymorphisms within each transcript.

We typically use paired end sequencing for RNA seq as it makes mapping much more straight-forward (although it's not essential). It basically gives us a lower number of reads that we have to throw out due to multiple mapping.

[Basia]

poly A purification from tomato total RNA

Hi all
I have been trying to get good mRNA from tomato total RNA using Dynabeads® Oligo(dT) from invitrogen. mRNA I am getting is a good quality with 1-2% rRNA contamination. My problem is the amount. I can only purified 0.2%of mRNA from my total RNA. Have anyone isolate mRNA from tomato? How much of poly A can I expect to be purified? What methods would you recommended to use.
Thanks,

[james hadfield]

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?

[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.

[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

[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

[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.

[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.

[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...

[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]

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