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
- - - - -

Comparing Gene Expression of Different Genes Using Semi-Quantitative PCR


  • Please log in to reply
2 replies to this topic

#1 lilac

lilac

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 17 March 2011 - 05:15 PM

Is it possibly to compare gene expression of different genes within a single sample using semi-quantitative RT-PCR? For example, if I ran an identical number of cycles for the same DNA sample with primers for three different genes, and then ran the PCR product on an ethidium bromide gel, would this reflect their relative gene expressions (aka, if one band for one gene were brighter than another band for another gene?) Or is it impossible to make this deduction?

I'm sorry if this is a bad question, I was just curious how exactly this would work!

#2 Nephrite

Nephrite

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 75 posts
3
Neutral

Posted 18 March 2011 - 01:17 AM

Is it possibly to compare gene expression of different genes within a single sample using semi-quantitative RT-PCR? For example, if I ran an identical number of cycles for the same DNA sample with primers for three different genes, and then ran the PCR product on an ethidium bromide gel, would this reflect their relative gene expressions (aka, if one band for one gene were brighter than another band for another gene?) Or is it impossible to make this deduction?

I'm sorry if this is a bad question, I was just curious how exactly this would work!


Hi lilac.

I am not competent at all, but basically I have the same task - to compare gene expressions.

Why don`t you have a look on these two papers, section Material and Methods?

Boonmars T, Wu Z, Nagano I, Takahashi Y. 2004 Parasitology, 128, 323-332
Wu Z, Nagano T, Boonmars T, Takahashi Y. 2005 Parasitology, 131, 373-381

Maybe they will give you some answers.

Nephrit

#3 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,188 posts
106
Excellent

Posted 19 March 2011 - 04:55 AM

I'm affraid it's not very accurate. Standard PCR only shows you state of the reaction on the end of the run. But at that time two differently expressed samples could have reached the plateau phase and you can't distignuish between them. Trying different number of cycles could help you to catch the phase where the reaction is not yet at plateau, but that would only help when you try to roughly compare between two samples of the same gene, whose expression in not to far away and not to close at the same time. Trying to compare two different genes is even more inaccurate, because different primers have different efficiency. You need to go real-time and do absolute quantification, normalise to houskeeping and assess efficiency.

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.