I have been doing a lot of total RNA extraction from wild type zebrafish. However, with other experiments going on simultaneously, I often find very little time left for dissecting the fish and extracting RNA. This also means that the fish are kept for longer than needed in the aquaria, which in turn means we are paying for them up until they are taken off the system.
Given that 4% PFA is successfully used for in situ hybridisation, can I fix my adult fish in 4% PFA and then rinse them with DEPC water just before dissecting them for RNA extraction?
Any advice please?
I would not recommend storing them in PFA if you're going to extract RNA. Try storing your samples in RNAlater. That will keep your RNA stable for very long periods stored at 4° or even room temperature. Alternatively, you could homogenize samples in Trizol and storing them at -80° for up to a few months. But I highly recommend RNAlater.
Our lab works with Zebrafish and Japanese medaka, and here is one method we use that preserves RNA quality and yield:
1. Euthanize fish in Tricaine.
2. Transfer fish to 1.5 ml microfuge tube (USA Scientific Seal-Rite) and remove excess tricaine/media.
3. Add up to 1 mL of TRI Reagent or TRIzol
5. Homogenize tissue using Biovortexer (FisherScientific #NC0400960) with attached pestle (USA Scientific #1415-5390). These pestles fit the 1.5 ml tubes and ensure optimal homogenization. Each sample takes 3-6x 10 second pulses. Use a new pestle for each sample OR wash thoroughly with ethanol and DEPC water.
6. Store homogenized samples at -80C for up to 3-4 months
***Be sure to use RNase spray to disinfect gloves, counter-tops, instruments, pipets etc to reduce the probability of contaminating samples.**
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