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 Extraction using Trizol


  • Please log in to reply
5 replies to this topic

#1 kbolt

kbolt

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 21 January 2005 - 06:33 AM

I am wanting to get the best quality and quantity of RNA possible. I have a Trizol protocol that states to use about 10 ul Trizol / 1 mg of tissue, and I am wondering if this holds true for brain tissue or if this is a rule of thumb for peripheral tissue. The tissue sample I have is smaller than 1 mg (it's just a punch of the rat brain). A girl in my lab has extracted RNA from the same type of brain tissue and used 250 ul Trizol for the same small amount of tissue (obviously disreguarding the 10ul/1mg). She successfully extracted RNA but it wasn't very high quality and quantity. I am sure there are many reasons for her results, but I am just brainstorming the optimal Trizol amount currently. So my question is...how much Trizol should I use for rat brain tissue? If anyone can give me a little guidance I would appreciate it!

#2 suhasbn

suhasbn

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 21 January 2005 - 08:24 AM

just stick to the manufacturers protocol (trizol) it shud work fine. good luk

#3 badcell

badcell

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 94 posts
1
Neutral

Posted 21 January 2005 - 10:35 AM

With Trizol, I believe that the more quantity of the reagent you use, the better the results you get. I work with small tissue samples and always use 1 ml Trizol. Don't forget you need to add a carrier such as glycogen in the isopropyl precipitation step.
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)

#4 kbolt

kbolt

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 21 January 2005 - 12:18 PM

With Trizol, I believe that the more quantity of the reagent you use, the better the results you get. I work with small tissue samples and always use 1 ml Trizol. Don't forget you need to add a carrier such as glycogen in the isopropyl precipitation step.

Thanks for the help! One more thing...glycogen isn't in my protocol. How much glycogen should I use in the isopropyl precipitation step...and what does it do exactly?

#5 pcrman

pcrman

    Epigenetist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,165 posts
68
Excellent

Posted 21 January 2005 - 03:17 PM

Glycogen is a carrier for the precipitation of nucleic acids (DNA or RNA). It is not a must for RNA extraction with trizol. I've never used trizol but used a similar reagent called TriReagent. I found it unnecessary using glycogen with Trireagent.

You can use 20 ug per sample. That amount will allow you to precipitate pg-amount of RNA from a volume of 1 ml.

#6 badcell

badcell

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 94 posts
1
Neutral

Posted 24 January 2005 - 07:32 AM

Glycogen is a carrier for the precipitation of nucleic acids (DNA or RNA). It is not a must for RNA extraction with trizol.


Yeah, you don't need to add glycogen if your sample is big enough, but I found that glycogen is necessary when working with small samples of tissue or cells. It increases a lot the quantity of RNA you can extract. I usually work with as few as 15000 cells and I got enough RNA to measure OD, run in an gel and do RT. I would certainly add glycogen for a sample of 1 mg tissue.
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)




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

©1999-2013 Protocol Online, All rights reserved.