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-   -   mRNA preparation for RNA-Seq (http://seqanswers.com/forums/showthread.php?t=1026)

Eshchar 01-12-2009 01:53 AM

mRNA preparation for RNA-Seq
 
Hi all,

Regarding RNA preparation for mRNA seq (we are using Illumina), we are using Qiagen's Oligotex kit for poly-T selection. So far we have observed that in the final sequencing, approximately 40% of our short reads are ribosomal RNA derived.

Can people perhaps share how much rRNA sequence is present in their Illumina RNA seq runs, and what sort of mRNA selection are they using?

Also, are any users familiar with the of Ribo-minus rRNA depletion kit from Invitrogen?

doxologist 01-12-2009 10:02 AM

we have some experience with Ribominus. However, your mRNA quality needs to be good for good removal. Fragments of rRNA without the consensus sequences will not be removed.

Melissa 01-12-2009 08:08 PM

Quote:

Originally Posted by Eshchar (Post 2899)
Hi all,

Regarding RNA preparation for mRNA seq (we are using Illumina), we are using Qiagen's Oligotex kit for poly-T selection. So far we have observed that in the final sequencing, approximately 40% of our short reads are ribosomal RNA derived.

Can people perhaps share how much rRNA sequence is present in their Illumina RNA seq runs, and what sort of mRNA selection are they using?

Also, are any users familiar with the of Ribo-minus rRNA depletion kit from Invitrogen?

I have tried Oligotex kit but not satisfied with the quality of cDNA synthesized (probably due to degradation). Have you tried two rounds of oligo dT priming using Dynabeads mRNA purification kit as recommended in the mRNA-seq protocol?

Check out Brian's reply at the following link http://groups.google.com/group/solex...9ff861ee6089a2.

Hope this helps.

monad 01-26-2009 11:34 AM

We've been dong several hundreds of mRNA-seq protocol using Dynabeads with extremely consistant and high quality output over last 16 months. Dynabeads is now packaged in Illumina mRNA-seq kit.

Eshchar 02-12-2009 11:46 PM

Thank you all for the advice, we have ordered the newer version of the mRNA-Seq kit from Illumina. I think we'll try that first and then change one thing at a time if it is still not good enough.

doxologist 02-13-2009 07:36 AM

The illumina kit has worked good in our hands... with little ribosomal content.

ieuanclay 06-04-2009 02:44 AM

We tried the ribominus kit and it worked well - I would agree with doxologist though, you need good quality! It is a good alterantive if you want to remove rRNA whilst not avoiding not polyA'd RNAs (small RNAs etc...)

Ieuan

wiggy 08-01-2009 02:16 PM

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.

walle 09-24-2009 11:32 PM

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

Eshchar 09-24-2009 11:56 PM

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

wiggy 09-27-2009 06:02 PM

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.

chameleon 07-27-2010 10:51 AM

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!!

NextGenSeq 07-27-2010 12:48 PM

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.

arabidopsis 01-13-2011 02:11 AM

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.

irit 02-01-2011 09:27 AM

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 02-02-2011 01:01 AM

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 02-02-2011 11:07 AM

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 02-04-2011 08:03 AM

Quote:

Originally Posted by li zhang (Post 34104)
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 02-04-2011 10:12 PM

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 02-07-2011 03:49 AM

Quote:

Originally Posted by li zhang (Post 34294)
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.


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