Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

RNA isolation -what is happening?-


  • Please log in to reply
5 replies to this topic

#1 vuutje

vuutje

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 26 March 2009 - 05:39 AM

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! :)

#2 Sciurus

Sciurus

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 26 March 2009 - 07:28 AM

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

http://en.wikipedia....form_extraction

Edited by Sciurus, 26 March 2009 - 07:28 AM.


#3 vuutje

vuutje

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 27 March 2009 - 02:36 AM

Thanks! I totally forgot wikipedia :)



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

#4 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,113 posts
59
Excellent

Posted 27 March 2009 - 09:07 AM

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.

#5 Andrzej

Andrzej

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 15 May 2009 - 04:13 AM

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

#6 wuxx0153

wuxx0153

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 35 posts
1
Neutral

Posted 15 May 2009 - 09:50 AM

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.

Edited by wuxx0153, 15 May 2009 - 10:01 AM.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.