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 with low number of cells (1*10^4 to 5*10^4) for RT-PCR


  • Please log in to reply
3 replies to this topic

#1 Wek

Wek

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
2
Neutral

Posted 11 December 2012 - 10:41 AM

Does anyone have any advice on how to optimally do this? I am trying to isolate the RNA but every time I do this my yield is very low. One obvious option would be to use more cells but that's not possible. I am using ReliaPrep™ RNA Cell Miniprep System but if anyone has a better suggestion I am all ears.

PS. Isolation is not the problem. I am looking for specific advice on how to isolate RNA from this small number of cells.

#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
109
Excellent

Posted 11 December 2012 - 11:01 AM

Column methods have the disadvantage that you can't lower the elution volume to concentrate your sample. You can try to elute the membrane twice (or more?) with the eluate to get more molecules from mebrane to solution. But the concentration would be always very low. You may also try to elute several times with a plenty of eluate and then concentrate the sample to get higher concentration, the yield in column methods depends also on elution volume.

I was isolating RNA from small colonies (100-1000 cells) with Trizol, but that has a drawbacks too. First it will always precipitate badly in low concentration so even when reducing the Trizol volume, you need to use carrier to help precipitation and some protocol modifications. Then you are washing the pelet several times, but the pelet is not visible, that is bit frustrating task. At the end you can dissolve the (invisible) pelet in very small amount of water to get reasonable concentration, but it would be still quite contaminated by trace phenolic compounds from Trizol (which are always there, but now you have much more RNA). Dipends if you care or not.
There are additional protocols how to clean it up by butanol or something like that, but not sure if it's adapted for lower yield samples.

Also if you do qPCR after that with a single set of genes, you may bypass isolation and use some Cells-to-Ct kits that lyse cells within PCR reaction, but that way you use the cells up of course.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 Wek

Wek

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
2
Neutral

Posted 11 December 2012 - 11:19 AM

Well, my cells will come fresh from a sort (flow cytometry) and I need to analyze about 10 genes. I will try to use an RNA carrier to see if it helps with the low yield. I am trying to stay away from phenolic compounds and concentrating the samples to avoid contamination and RNA degradation. By any chance do you have any recommendation for a RNA isolation kit design for low number of cells?

#4 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
109
Excellent

Posted 13 December 2012 - 12:27 PM

It helps definitelly with the precipitaion methods. Probably not with the column methods, but I'm not sure. Some people mention using prewarmed water for elution (55 deg) and longer time before spin (5 min). You may try to find better columns, if you're not limited by money much.
For example the top companies like QIAGEN of Ambion (former) may have solutions.
Like this one (didn't tried) though it's a bit extreme:
PicoPure RNA Isolation Kit

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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

©1999-2013 Protocol Online, All rights reserved.