This material is from the 4th edition of The Zebrafish Book. The 5th edition is available
and within the
ZFIN Protocol Wiki
CHAPTER 9 - MOLECULAR METHODS
Rapid Isolation of RNA from Zebrafish
(Source: M. Westerfield)
This is a modification of the method of Chirgwin et al. (
:5294-5299, 1979) which can be used to obtain RNA from zebrafish embryos or adults for northern analysis, etc.
1. Homogenize embryos (or adults frozen in liquid N
) in GHCl buffer with 0.5% diethylpyrocarbonate (DEPC) added. Use 0.5 ml GHCl buffer per 100 embryos, or 2 ml per adult.
7.5 M guanidinium hydrochloride
0.025 M NaOAc pH 7.0
5 mM dithiothreitol, 0.5% N-laurylsarcosinate.
2. Extract once with an equal volume of phenol-chloroform.
3. Transfer the aqueous (upper) phase into a new tube and precipitate the RNA by adding 2 µl of 1 M acetic acid and 50 µl ethanol for each 100 µl of GHCl solution. Incubate at -20°C for at least 3 hr.
4. Pellet the RNA by centrifugation for 5 min, 10k.
5. Remove the supernatant as completely as possible and redissolve the pellet in half the original volume (but minimally 50 µl) GHCl buffer (without DEPC).
6. Reprecipitate the RNA as in step 3.
7. Wash (and store) the RNA pellet in 100% ethanol. For northern analysis the RNA is best dissolved directly in northern sample buffer.
The Zebrafish Book