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
- - - - -

No RNA after Trizol extraction


  • Please log in to reply
8 replies to this topic

#1 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 02 December 2009 - 02:40 AM

I carried out RNA extraction from mango fruit pulp using Trizol reagent (Invitrogen) exactly following the protocol. Although there was a good (too large) pellet after isopropanol precipitation my gel was blank! What could be the reason?Is there pellet because of polysaccharide contamination? In the absorption spectra, maxima was at 230 and not at 260! Is this because of phenol/thicynate contamination as I have read somewhere?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#2 gogreen

gogreen

    Somewhere I belong

  • Active Members
  • PipPipPipPipPip
  • 90 posts
0
Neutral

Posted 02 December 2009 - 05:11 AM

Hey Ram!

I am not sure, how much of RNA to expect from Mango fruit pulp, but I guess it should not be much...its quite possible that you are actually getting the polysacharides from the solution. I once tried isolating RNA from banana leaves and always used to end up with perfect looking huge pellet, but this never yielded to dissolve completely and my quantification were grossly wrong possibly because of the huge amounts of polysacharides in the leaves...Try adding more of chloroform maybe 1ml: 300- 350ul against 1ml:200 ul..This might help

#3 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 08 December 2009 - 10:57 PM

Now I carried out RNA extraction from mango leaf (sample 1: ~100mg, sample 2: <50mg) using Trizol. Included additional chloroform wash before isopropanol precipitation. Aqueous phase after Trizol separation was brownish (may be because I used brown-coloured tender leaves). This phase turned turbid after isopropanol addition and gave good pellet upon centrifugation. After loading the sample on gel, I did not see anything. Its completely blank!! Absorbance spectra is something like this..Please help!! :lol:


Posted Image
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#4 gogreen

gogreen

    Somewhere I belong

  • Active Members
  • PipPipPipPipPip
  • 90 posts
0
Neutral

Posted 09 December 2009 - 01:31 AM

Once you homogenize the tissue, leave it in trizol for 5-10 minutes, spin at > 10k rpm/g and take the supernatant and then proceed with the chloroform...I hope you got rid of the tissue debris this way...As I said in the last post, the pellet what you see might not be RNA eventhough u see a perfect white pellet!

Trizol might not work for every plants..it didnt work for banana in my case, but worked for leaves and flowers of maize, sunflower, rice, barley, sorghum and even watermelon fruit...

the brown/greenish color that you see is normal..the color remains even in the pellet sometimes, but i've used it for microarray experiments successfully

#5 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 09 December 2009 - 01:45 AM

I have tried separating tissue debris after trizol mixing...that too didn't work! Do you get complete absence of RNA? I should have got at least some RNA..may be very faint bands ! But absolutely nothing is there...I wonder WHY!!
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#6 gogreen

gogreen

    Somewhere I belong

  • Active Members
  • PipPipPipPipPip
  • 90 posts
0
Neutral

Posted 09 December 2009 - 02:36 AM

Its been over a year since I did these experiments..but I vaguely remember that the bioanalyzer profiles of these samples showed no RNA in my case too! After trying almost every method possible, I moved to a much laborious hot borate extraction protocol. I searched the protocol in the net and found these 2 versions...it is the same protocol that i had used..


Did u try with some other method already??

Attached Files


Edited by gogreen, 09 December 2009 - 02:43 AM.


#7 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 14 December 2009 - 06:19 AM

Thanks for the info! Yes gogreen, we have already tried CTAB method which very well. But its quite laborious; since I have many samples, was trying to find out a simple method!
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#8 gogreen

gogreen

    Somewhere I belong

  • Active Members
  • PipPipPipPipPip
  • 90 posts
0
Neutral

Posted 14 December 2009 - 09:47 AM

All the best...Hope u succeed in finding a better option..do update if u come across one..I'd spent quite some time doing over 200 extractions with that protocol :lol:

#9 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 21 December 2009 - 11:59 PM

Hi...Got good yield of RNA from mango fruit pulp with some additions in the Trizol protocol
75 mg tissue + 0.75ml extraction buffer
[100 mM Tris-Cl (pH 8.2),1.5 M NaCl, 30 mM EDTA (pH 8.0), 2% CTAB and b-mercaptoethanol]
Incubated 65C for 20 min--cooled + 0.5ml trizol
Rest everything by Trizol protocol with inclusion of additional chloroform wash before isopropanol precipitation..
Got 300ng/ul RNA in the volume of 50 ul. There was very small genomic DNA contamination that could be removed by DNase treatment

Edited by ram, 21 December 2009 - 11:59 PM.

If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.




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

©1999-2013 Protocol Online, All rights reserved.