ribo-zero magnetic kit

[marRoma]

hi!

I have a question about the way to purify the rRna-depleted RNA , after the ribo-zero magnetic kit. the ribozero kit suggests either the ethanol precipitation or the rneasy cleanup kit or agenancourt RNAclean but i have the rna clean and concentrator kit by zymo research; can i use it istead of that one suggested by epicentre? does anyone have experience with this kit? because i'd like to avoid the ethanol precipitation, i've read that the concentration of RNA decreases a lot.

thank you

Margherita

[nucacidhunter]

We use RNAClean knowing that it is poor in recovering small RNA fraction which we are not interested. Methods recommended by Epicentre have been tested for various properties. One can use similar product, but it is unknown if they introduce any bias. Column based cleaning products normally have different size bias (some are poor in recovering large fragments) which may or may not be crucial for downstream applications. So, it is best to use recommended products to be safe.

[kerplunk412]

I actually tested RNA cleanup methods about a year and a half ago. Ethanol precipitation was the highest yielding method, but it does take a long time and there is a lot of chance for loss when dealing with tiny pellets. The Zymo C&C worked well in my hands, and I even changed the lab RNA-Seq protocol to use it instead of RNEasy Minelute. If you use the RNEasy kit it is very important that you use the Ribozero protocol, or more specifically the amount of ethanol indicated in the Ribozero protocol. In my experience the yield from the Minelute kit is not very good unless you use much more ethanol than indicated in the manual. The Zymo C&C kit worked well with the volume of ethanol indicated in its manual, but I do recommend re-applying the eluate to the column at least once for increased yield, especially if eluting in a small volume.