ZF RNA Extraction

Chi-Bin Chien chi-bin.chien at neuro.utah.edu
Wed Apr 6 11:02:00 EST 2005


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I would echo Melissa Haendel by saying that we routinely use Trizol  and
it works well. We usually do 10-100 embryos or larvae, but have  done
single-embryo analysis as well (in cases where we needed to
PCR-genotype individual embryos). I have never dechorionated or used  an
electric homogenizer--we just triturate through a small-bore
hypodermic needle (sometimes a bigger one first then a smaller one), 
which works well to disrupt the tissue.

I collect the embryos in a single tube, then quickly remove excess 
medium, add Trizol, and triturate. The key is to break open the cells 
quickly so that the guanidinium in the Trizol can inactivate RNAses 
before they start attacking your RNA.

Chi-Bin Chien

Mark asked:

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>
>Hi again all,
>
>Could anyone advise me on a protocol for extracting RNA from zebrafish
embryos.
>
>I am using the small-scale SIGMA TRIZOL reagent protocol and i am getting
very poor results.  I usually de-corionate then isolate the RNA in
batches of ~20 embryos (0-24 hrs) and batches of 10 for 48 (hrs).
>
>The yields are very poor, and the quality (in terms of ibosomal RNA) is
also poor.
>
>If anyone uses this method or another method of RNA extraction i would
like to hear from them.
>
>Cheers
>
>Mark
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