I am preparing a methanol stock of fixed embryos at different developmental stages (up to 5dpf) and storing them at -20C until further use. I fix embryos in 4% PFA in DEPC-PBS at 4C over night, then wash them twice in DEPC-PBT and perform methanol series (25%-absolute). In the end I rinse the embryos in absolute methanol again and store them in 2ml epi at -20C.
Can I use the embryos that received this treatment also for qPCR? I am worried about RNA quality after fixation and dehydration.
The situation is, that I'm getting a large batch of embryos and I need to fix them at certain developmental stages, but this is not possible in my own lab, so I'll be unable to extract RNA straight after euthanasia.
Does anyone have any suggestions?
Thank you!
Hi Monika,
For RNA extraction I would always snap freeze tissue directly in liquid nitrogen and store at -80oC until the time of extraction. This would be the best way to avoid any RNA degradation. If you are using something like TRIzol (http://www.lifetechnologies.com/order/catalog/product/15596026) for your RNA isolation you can also freeze your samples directly in this and continue the isolation protocol later.
I hope that helps!
Beck
Dear Monika, I fully agree with Dr Richardson. I personally collect the embryos at the desired stage, I remove the fish water (embryo medium) and rinse them with 1x PBS, then I just add Trizol and store at -80 C until the time of RNA extraction. Obtained RNA was always of excellent quality after Trizol protocol, in my hands. Good luck, Natascia
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