Does anyone routinely do RNA isolation from zebrafish embryos?
I have embryos from different stages but all below 24hpf. This is the protocol I follow:
- Take 10-20 embryos
- Wash once with milliQ water
- Treat with 1mg/ml Proteinase-K (5min with gentle shaking) to dechorionate
- Wash again with milliQ water
- Remove water and add 250 µl Trizol
- Homogenize by pippeting
- add 750µl more of trizol
- Do the standard trizol extraction step (with chlorofom)
- Take the aqueous phase and wash again with 200µl chloroform
- Precipitate RNA with isopropanol
- Wash thrice with 70% ethanol
- dissolve in nuclease free water
Despite all this I get a poor A260/A230 : ~0.4
Moreover, this happens consistently in all samples. The RNA bands look fine on the gel.
I have done RNA isolation many times from cell lines and tissues and never faced this problem. Is this common with zebrafish embryos? Is there a way to fix this other than by using kits?