mRNA preparation for RNA-Seq

[ieuanclay]

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

[doxologist]

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

[Eshchar]

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.

[monad]

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.

[Melissa]

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.

[doxologist]

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.

[Eshchar]

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?