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

Extraction and Purification of RNA from Zebrafish Embryos

(Source: A. Molven)

1. Homogenize 3 ml of zebrafish embryos (approximately 3000 embryos) in 20 ml of GIT buffer. Use an automatic tissue homogenizer (e.g. Dounce) and homogenize until all the embryos are totally disrupted (2-3 min).

GIT buffer:

4 M guanidinium isothiocyanate
0.1 M Tris-HCl pH 7.5
1% ß-mercaptoethanol.

2. Add N-laurylsarcosinate to 0.5% and mix. Transfer the homogenate to two 15 ml Corex tubes, centrifuge 5k, 5 min (HB4 rotor) to sediment chorions and cell debris.

3. In ultracentrifuge tubes, layer 0.3 ml of GIT buffer onto a pad of 3.3 ml, 5.7 M CsCl in 50 mM EDTA, pH 8.0. On top, carefully layer 7.1 ml of the homogenate.

4. Centrifuge in a swinging bucket rotor (e.g. SW-41) for 20 hr, 30k, 20°C.

5. Carefully remove the supernatant without introducing any exogenous RNase contamination. The RNA should now be visible as a glassy pellet.

6. Dissolve the pellet in 0.5 ml TES buffer (1% SDS 5 mM EDTA, 10 mM Tris-HCl pH 7.4) and transfer to a nuclease-free 15 ml Corex tube.

7. Heat to 65°C to ensure dissolution of the pellet. Washing the bottom of the ultracentrifuge tube with another 0.5 ml TES buffer will enhance the recovery of RNA.

8. Determine the concentration of RNA and precipitate by adding 0.1 volume of 2 M sodium acetate, pH 7, and 2.5 volumes of ethanol.

9. Store the RNA at -20°C.

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