Best method to mRNA purification

[matiasfreired]

Hello everyone
I'm looking for the better method to purify mRNA to use in RNAseq. I studied the Ribozero method from Illumina and Ribominus from Ambion, but both methods are very expensive in comparation with Mag-Bind mRNA Kit of Omega Biotek, based on Olido(dT) beads.
Did anyone use this method to isolate mRNA before?
The mRNA quality is appropiate to RNAseq?
Please, could you share your experience?

[cascoamarillo]

There's no best method, but most appropiate. It depends what you really wanna look for: mRNA or RNA transcripts in general (codifiying or not). PolyA selection kits work great, but remember you only select mRNA with polyA tail.
On the other hand, Ribozero/Ribominus methologies look good, though is your species suitable for that? Would its oligos work on your rRNA?
In general, RNAs coming from polyA kits or ribozero should work for RNAseq. Depending on how much RNA as an input you use and output you obtain from these kits, maybe you have to choose different protocols-approach. Nowadays there are RNAseq library prep kits which allow really low amount of RNA as input (it depends how much do you want to spend).

[SS00]

I ran a Poly A Prep on Total RNA and found that even with Poly A selection I still had reads that were mapped as ribosomal. This was really curious, but I guess some rRNA's are polyA?

However, this was my first RNA-seq experiment and I will soon be following up by comparing Total, Poly A, and both methods with Ribo Depleted RNA (with Ribo Zero).

[matiasfreired]

We want to determine differential expression, so we are thinking on mRNA sequencing. We can obtain a good amount of total RNA, so the total RNA no represent a problem to us.
And, respect to purification with PoliA column, we have the same trouble with a PolyA purification, it's common a contamination with rRNA with this method.

[Michael.Ante]

I ran a Poly A Prep on Total RNA and found that even with Poly A selection I still had reads that were mapped as ribosomal. This was really curious, but I guess some rRNA's are polyA?

In my experience, you have to distinguish between mitochondrial and nuclear rRNA. Mitochondrial rRNA has an intrinsic polyA tail, whereas nuclear rRNA doesn't.

[ojham]

Hello , I need to eliminate ribosome sequence from my Vitis Riapria RNA sequence . Can anyone help me where can I find ribosomal database database for V. riparia ? It would be great help . Thank you..

[Birdman]

matiasfreired For RNAseq, I used Poly(A)Purist™ Kits from Ambion with great success, and if you have a lot of sample, I found it quite fast to do. This kit will obviously select polyA RNAs.

[Birdman]

ojham Although I did not use it, I believe you could use RNAmmer for that.

[ojham]

Thank you . I will try and let you know if it worked .

[ojham]

I could not find the data