Hey Sanna,

I used to extract RNA from 12 x 3dpf old embryos and would typically get 300-400ng/µl. I have extracted using Trizol followed by an on-column purification (using the Qiagen RNeasy micro kit) but I have also done it purely with the micro kit (instead of trizol, you use RLT buffer which is inlcuded in the kit). In my experience it is easier to use the RLT buffer as 1) there is no need to keep your sample on ice during any of the steps and 2) there is no need to do a phenol:chloroform extraction step and therefore there is no chance of phenol contamination (which you would notice when reading your samples on the nanodrop). Using the micro kit, I almost always get perfect ratios on the nanordop (260/280 = 1.8-2 and 260/230 =  2-2.1) and good quality RNA when testing on the bioanalyzer (RIN >8) - which is ideal for sequencing. 

Hope this helps,

Andrew

Hi Andrew! Thank you so much for that answer - I will follow your advice. Did I understand you correctly that you get 300-400ng/ul when using trizol protocol? (what volyme do you dilute in? whats the total amount?). Is the yield as good when you use the RNeasy micro kit? Do you normally go for the "Purification of Total RNA from Animal and Human Tissues" protocol in the Qiagen kit? 

And one more question: What is your experience with freezing the embryos (-80)? Does that affect the yield/quality of the RNA? 

Hey,

I got the same amount using both methods - I eluted in 14µl of RNase free water included in the micro kit meaning the total amount was between 3.6-4.8µg of RNA (there is a 'dead space' when eluting from a column, meaning that when eluting with 14µl you can expect to recover ~ 12µl).

Yes that is the protocol I use. Don't forget to add Beta Mercaptoethanol or DTT to your RLT buffer (if this is what you want to use) - this will inactivate RNases and is therefore important.

Freezing embryos is good...you just have to take care about how you do it. My suggestion would be to place your embryos immediately in a commercially available solution to inactivate RNases (e.g RNAlater) then store at 4 degrees or -20 or -80 (depends how long you wait before extracting). Then on the day of the extraction, remove the solution, add Trizol or RLT buffer, homogenize your embryos and then extract. If you freeze your embryos in trizol or RLT buffer, you can precipitate salts which can be tricky to remove during extraction and can give you poor ratios on the nanodrop.

Thank you Andrew! This is all really useful information. Im very grateful you took the time to answer. Have a great weekend!

Best regards,

Sanna

No problem Sanna. Good Luck!

Hi all,

Thank you very much for you input. After your suggestions and others this is what I end up doing, and i worked very well:

I extracted from pools of 10, 20 and 40 3dpf embryos and got around 250 ng RNA/fish and a RIN of 10 for all samples. 

BR

Sanna

Hi!

I actually have a question for Andrew Lumley, regarding the RNeasy micro kit. In your first answer you stated "but I have also done it purely with the micro kit (instead of trizol, you use RLT buffer which is inlcuded in the kit)."

I want to use the kit for RNA extraction of 10 ZF heads/sample, and I wonder which homogenizing procedure you followed. Did you follow one of the kit's methods (and if so, which one) or another one?

Thank you!