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-   -   Substitute for Ribozero beads? (http://seqanswers.com/forums/showthread.php?t=64711)

luc 12-04-2015 03:23 PM

Substitute for Ribozero beads?
 
Has anybody had success in substituting the ribozero magnetic beads (which are the limiting reagent in the kits)?
They are very likely simple streptavidin coated beads; but how much of the substitute will be required?

Thanks in advance!

nucacidhunter 12-04-2015 07:24 PM

I do not have direct experience with your query. Beads have a binding capacity that can be found in manufacturer specification sheet and quantity or number of transcripts that are depleted can be estimated. Using twice the estimated amount is a good starting point.

On the subject of rRNA depletion there are other products which are more cost effective:

1- Lexogen RiboCop (works by hybridisation of RNA to probes and binding probes to beads similar to Ribo-Zero Gold)
2- RNaseH based degradation product from NEB
3- Clontech SMARTer products with kit included RiboGone (RNaseH based) reagent. These products have a considerable time-saving advantage as well because they have ligation-free workflow
4- Post-cDNA synthesis depletion workflows

luc 12-10-2015 08:58 PM

Thanks!

for plant RNAs the choices are fewer, though. I am looking to substitute the beads for another lab that has lost the part of the kit (the beads) but still has the reagents that were stored in the freezer.

This protocol (http://www.jove.com/video/50093/depl...enomic-rna-seq) uses NEB streptavidin beads and should be pretty easy to follow (I am not generating our own baits for the moment).

nucacidhunter 12-11-2015 02:00 AM

The issue would be the unknown buffer composition of the beads. They might have adjusted salt concentration to make it compatible with rRNA Binding Buffer carried over to beads. If the buffer is not right then it might affect binding and result in carry over of rRNA to next step. This applies to Ribo-Zero plant that comes with TruSeq stranded total RNA-Seq kit. The workflow for epicentre or stand-alone Ribo-Zero is different but the composition of Magnetic Bead Resuspension Solution still is unknown.

Edit: for plants other option is NuGEN Ovation kits that uses In-DAC technology and they can design custom probes but I am not sure if hey charge more for custom probes. They mention that design is free.


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