RNA EXTRACTION FROM ZEBRAFISH EMBRYOS

By Anat Yarden and Sigal Bitzur

Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

We have recently modified a LiCl RNA extraction protocol (Auffray and Rougeon, Eur. J. Biochem. 107:303-314, 1980), to suite our needs in RNA extraction from ZF embryos. With the protocol described below we have routinely obtained approximately 10 micrograms/ 60 ZF embryos. The procedure is simple and convenient, as all steps are carried out in microfuge tubes.

1. Collect about 60 ZF embryos in sterile microfuge tube and remove as much liquid as possible.

2. Add 0.8 ml Sol. I (3 M LiCl, 6 M UREA, 0.1% SDS, 10 mM NaAc pH 5.5, 200 U/ml Heparin) and homogenize the embryos using a syringe and a 21 g needle 10 times. Keep on ice for at least 16 hrs (in the cold room).

3. Spin in microfuge for 15 min. at 4 C, resuspend pellet in 1 ml Sol. II (4 M LiCl, 8 M Urea) and spin again. Repeat step 3.

4. Resuspend pellet in 0.2 ml Sol. III (0.1 M NaAc pH 5.5, 0.1% SDS), extract twice with an equal volume of phenol:chloroform (1:1), and once with chloroform. Precipitate with 70% ethanol and 0.3 M NaAc.

5. Resuspend the RNA pellet in water and determine the OD. We have used this RNA directly for Northern analysis.


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