RNA isolation -what is happening?- - (Mar/26/2009 )

Hi all,

I'm a total newbie here and with RNA isolation. I got a protocol and it is working really well...
BUT I'm doing al these different steps and I have NO idea what is happening inside the tube :rolleyes:
Could someone explain to me what which step is doing?

I first grind my tissue with the use of Trizol, after that I add chloroform, vortex and spin for 10 minutes.
After that I take of the supernatant (so something is seperated from something else.. and there is also a white layer between the two phases, is that protein or?). To that supernatant I add isopropanol, which I leave for 30 minutes at RT and after that another 30 minutes of spinning. Which gives me in some way a pellet with my RNA. And after washing with EtOH (to take away the isopropanol?) I air dry my pellets and store them.

I really like doing RNA isolations etc. but I don't like the fact that I can't see whats going on inside the tube.
And its really frustrating if you can't see something happening AND don't know what is going on

It would be great if someone here can tell me whats going on or give a link to another site with usefull information.

Thanks!

-vuutje-

The only information I have off the top of my head right now is good ol' wikipedia :rolleyes:

http://en.wikipedia.org/wiki/Guanidinium_t...form_extraction

-Sciurus-

Thanks! I totally forgot wikipedia

*just in case people wonder... YES I'm blonde *

-vuutje-

well first of all I would have to say blonds are hot!

second, I am also new to RNA isolation but I read that you leave your sample and Iso-p for 30 min at RT?...30 min is ok but I usually leave for 5 min......also 10 min centrifuge at highspeed is sufficient.

don't you add anything to your air-dried sample before putting at -80? you need to add RNase free water or other buffers.

-Curtis-

you don't sound too blonde to me at all. Just the mere fact that you actually are interested what is going on in your tube, rather than just blindly following the protocol, suggests your brain cells are far from blonde
cheers!
Andrzej

-Andrzej-

vuutje on Mar 26 2009, 06:39 AM said:

I first grind my tissue with the use of Trizol, after that I add chloroform, vortex and spin for 10 minutes.
After that I take of the supernatant (so something is seperated from something else.. and there is also a white layer between the two phases, is that protein or?). To that supernatant I add isopropanol, which I leave for 30 minutes at RT and after that another 30 minutes of spinning. Which gives me in some way a pellet with my RNA. And after washing with EtOH (to take away the isopropanol?) I air dry my pellets and store them.

It also dependent what tissue you using and how you do it.

Some tissues naturally contain lots of RNase, such as pancreas is a pain in the behind.
If you encounter those tissues, single TRIzol extraction is not enough for RNA protection.

For how you do it:
First, for tissue application, 100mg tissue/ml TRIzol is the minimum (at least for me), and over that you risk insufficient protection.
Second, how fast you grind your tissue, the longer you take the less protection your RNA will have.

-wuxx0153-