This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.
CHAPTER 9 - MOLECULAR METHODS
Preparation of High Quality RNA from Zebrafish Embryos
(Source: S. Schulte-Merker and C. Nüsslein-Volhard)
This procedure, which is adapted from Brown and Kafatos (J. Mol.
Biol. 203:425-437), produces high yields and is fast (from
embryo to EtOH in less than 2.5 hours). Also, it works well for all stages and
tissues and yields high quality RNA. One drawback is the danger of working
with hot phenol; use extreme care to assure that the nitrogen has boiled off
1. Cool a mortar on dry ice; use liquid
nitrogen to cool other equip-
2. Pour embryos (100 or more) into a small
sieve and "dry" them by holding a paper
towel against the sieve (or towel dry adult).
Transfer the embryos (or adult) to the
mortar containing liquid
3. Homogenize the embryos (or the adult)
carefully with a precooled pestle while there
is still nitrogen present. This is best done by
pounding, not grinding, with the pestle.
When the liquid nitrogen is gone, grind
everything up to a fine
4. Transfer the powder to a Falcon tube (50ml)
containing liquid nitrogen. Use a precooled
spatula to transfer the powder as
5. The powder can be stored at -70°C for a couple of days.
Make sure that the powder never thaws
before proceeding to step
6. Heat a 1:1 mixture of unbuffered phenol
(pH 4-5) and 2xNETS (200 mM NaCl, 2
mM EDTA, 20 mM Tris, pH 7.5, 1% SDS)
to 95°C (at this
temperature, the mixture forms only a single
wear goggles, gloves,
7. Take the Falcon tubes from step 4/5 and let
the liquid nitrogen boil away completely.
8. Make sure the opening of the tube points
away from you. Immediately add about 10
ml of the 95°C hot
phenol/NETS mixture to the
9. Close the tube and vortex immediately for 1
minute. The RNA is safe now, and you can
do as many samples as you
10. After the samples have cooled, centrifuge
for 20 minutes at 5,000
11. Remove the supernatant and re-extract the
organic phase with 1 vol of 2xNETS.
12. Combine the two aqueous phases and extract
them once more with unbuffered
13. Centrifuge and extract the aqueous phase
twice with phenol:chloroform and once with
14. Precipitate the RNA by adding 0.1 vol 3 M
NaAC and 2.5 vol EtOH. Highest yields are obtained by leaving the samples
for a couple of days at -20°C.
15. After centrifugation, wash the RNA with
70% EtOH and dissolve in DEPC-treated
water. Typical yields are around 1 µg
RNA per embryo.
This method can be scaled down if RNA from only a few embryos is
required. In this case, collect embryos in an Eppendorf tube. Remove as much
water as possible and put the tubes into liquid nitrogen. With a precooled device,
grind the embryos to a fine powder. Afterwards, follow steps 4-15 and scale
down the required volumes accordingly.
The Zebrafish Book