Preparation Of High Quality RNA From Embryos

(Source: S. Schulte-Merker and C. Nüsslein-Volhard from Zebrafish Book 5th Edition)

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 tis¬sues and yields high quality RNA. One drawback is the danger of work¬ing 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 pound¬ing, not grinding, with the pestle. When the liquid nitrogen is gone, grind everything up to a fine powder.

4. Transfer the powder to a Fal¬con tube (50 ml) containing liquid nitro¬gen. 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 unbuffer¬ed 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 imme¬diately 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 volume of 2xNETS.

12. Combine the two aqueous phases and extract them once more with unbuffered phenol.

13. Centrifuge and extract the aque¬ous phase twice with phe¬nol:chloroform and once with chloro¬form.

14. Precipitate the RNA by add¬ing 0.1 volume 3 M NaAC and 2.5 volumes 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 embry¬os 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 re¬quired volumes accordingly.