So we are trying to collect a large amount of mouse embryos to do RIP-seq. I'm wondering whether I can freeze the embryos directly at -80 after each collection or I have to do the slow freeze/ fast thaw method which is normally used to cryopreservation/revive the embryos? Thanks.
Hi Jack,
I think your methodology would depends on what you’re expecting.
An RNA Immunoprecipitation coupled with sequencing (RIP-seq) to identify RNAs that are bound by an RBP of interest needs to be done on the classic controlled slow freezing / fast thawing procedure. Indeed, the uncontrolled slow freezing process (at -80°C) could leads to RNA degradation during the whole “cryopreservation” procedure (i.e., freezing, heating, thawing, and washing steps).
Moreover, did you considered the vitrification technique?
Hi Ludovic,
Thanks for the reply. I think all I need is just to maintain the protein-RNA interaction before I get enough embryos. I don't really need to revive the cells to produce more mice. Would the tissue culture freeze/thaw procedure work on the embryos as well? And the vitrification technique does seem to be a good idea and I have to look into that.
Hello Jack Wang,
I guess Vitrification step is most suitable and cost economic for your purpose.
Slow freezing needs the special equipment's and high quantity liquid nitrogen and time consuming process...as Ludovic told, yes there will be RNA degradation is seen in slow freezing protocols, so better u go for Vitrification procedure which will fetch u better results. And Vitrification media is commercially available in the market.
Regards,
Sanketh D.S
Hi Jack, Your objective is to identify RNAs. Although vitrification is quick and provides good results, however, I suggest to compare the procedure on vitrified and fresh embryos because vitrification alters RNAs.
Thanks Sanketh and Murid for the answers. I will try my best to get this experiment done as soon as possible to minimize the degradation. As to comparing vitrified and fresh embryos, even though it's a good idea and that's what we should do as a control, I don't think it's very feasible to us. It's quite hard to collect enough fresh embryos to do anything besides staining.
Hello Jack, I agree with Ludovic, Sanketh and Murid idea of using vitrification. May I know which stage of embryos are you focusing on?
- Badges
- Science topic
- Similar topics
- Computer Communications (Networks)
- Simulators