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 completely.

1. Cool a mortar on dry ice; use liquid nitrogen to cool other equip- ment.

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 nitrogen.

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 powder.

4. Transfer the powder to a Falcon tube (50ml) containing liquid nitrogen. Use a precooled spatula to transfer the powder as quantitatively as possible.

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 5.

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 phase). Beware: wear goggles, gloves, etc.

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 powder.

9. Close the tube and vortex immediately for 1 minute. The RNA is safe now, and you can do as many samples as you wish.

10. After the samples have cooled, centrifuge for 20 minutes at 5,000 rpm.

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 phenol.

13. Centrifuge and extract the aqueous phase twice with phenol:chloroform and once with chloroform.

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.

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