[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?

[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,

[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.

[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]

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]

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!

[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

[irit]

Have you tried running your ribo-depleted RNA on Bioanalyzer? You won't be able to get a proper concentration reading but you should be able to see the size distribution of your RNA.

[arabidopsis]

I used RiboMinus of Epicentre and got a weird result. RNA concentration after rRNA depletion was very low (predictable), but that low that I could hardly detect it by nanodrop. Does anyone know what rRNA depleted sample from HELA cells should look like (concentration, quality control values etc)? Can you detect RNA concentration after rRNA removal? I got about 20 ng/ul, but measurements are inconsistent and both 260:280 and 240/260 ratios are very low.

[NextGenSeq]

Epicentre sells a RiboZero kit. They also sell library kits which are much cheaper than ILMNs. We just got both of these but haven't tried them yet.

[chameleon]

rRNA depletion

Does anybody have experience for rRNA depletion from drosophila transcriptome?

Ribominus is said to be specific for human and mouse or yeast and bacteria. Is there anything that you may suggest?

Thanks!!

[wiggy]

My sequences were on ncbi so I just used it to design primers.

Or you could find highly conserved regions, create primers, sequence, and repeat until you get the whole sequence.

I believe this step is imperative. If you are using Poly A purification then any Rnase contamination or degradation before the last purification step will lead to sequences further from the A tail not being incorporated and thus not sequenced.

This is why we designed primers around the 5' end of the cdna.

[Eshchar]

Hi,

Our latest batch of libraries looks much better, with library preps only using the default Illumina mRNA-Seq kits (and whatever beads they contain). rRNA content seemed to be only around 2%-4% for each of the six libraries. Seems the problem was only in the old sample prep method before the dedicated RNA-Seq kits came out, and 2 rounds of mRNA selection using magnetic beads did the trick. Can't comment on Ribominus then, at the moment we're still only looking in poly-adenylated RNA.

Thanks again for the advice.
Eshchar

[walle]

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

[wiggy]

We subjected RNA to 2 rounds of PolyA purification from Promega and also to 1 round of Ribominus.

After analyzing via qRTPCR for both 18S and 28S rRNA we found the PolyA kit almost completely eliminated rRNA after 2 rounds.

Next we created primers for some large genes near the extreme 5' region. Some of these had been previously verified as low expression levels via more 3' primers. We found these transcripts to be represented in the polyA 2x.

Ribominus was not effective at all, perhaps because our rRNA's did not contain the consensus sequence.