poor RNA quality from zebrafish embryos

Question

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:

1. Take 10-20 embryos
2. Wash once with milliQ water
3. Treat with 1mg/ml Proteinase-K (5min with gentle shaking) to dechorionate
4. Wash again with milliQ water
5. Remove water and add 250 µl Trizol
6. Homogenize by pippeting
7. add 750µl more of trizol
8. Do the standard trizol extraction step (with chlorofom)
9. Take the aqueous phase and wash again with 200µl chloroform
10. Precipitate RNA with isopropanol
11. Wash thrice with 70% ethanol
12. 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?

Answer 1

Usually I had no problems with RNA extractions - from various cultured cells as well as a number of different tissues from mouse. If I look at your protocol this is pretty much the same as the one we used or the one recommended here for zebrafish embryo. This protocol "Purification of RNA Using TRIzol (TRI Reagent)" seems also interesting.

What can happen is a contamination by phenol or carbohydrates, then we usually used the protocol below to clean up the sample. You can also use column based methods, but they are more expensive.

• add 1/10 vol of NH4OAc (5M), 2.5 volume 100% cold EtOH (100%) and 10µg glycogen (for total RNA we usually didn't use any co-precipitation agents) and mix well
• incubate 30 min at -80°C
• centrifuge 20 min at 12000g at 4°C and remove the supernatant
• wash with 75% ice cold EtOH
• centrifuge 5 min at 12000 g at 4°C and remove supernatant
• Re-suspend the pellet in RNAse free water (can take some time)

Answer 2

This addition to the protocol worked out and good RNA quality was obtained:

Homogenization

• Homogenized the embryos, suspended (and frozen) in trizol by passing through 2ml insulin syringes. (About 10 times).

Washing

• Washed 2 times with 500µl 80% Ethanol
• Washed 3 times with 500µl 70% Ethanol

Dechorionation was dispensable and Proteinase-K treatment was too harsh on early stage embryos. So I did not dechorionate this time.

I think the number of washes can be reduced (1×80% and 2×70%) but this is what I stuck to (out of superstition :P)