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
- - - - -

Using DNA as a reference in qRT-PCR


  • Please log in to reply
1 reply to this topic

#1 Scienceboy

Scienceboy

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 13 April 2011 - 02:46 AM

Hi!

My plan is to make a qRT-PCR (using RNA) from soil sample to study the activation of one gene in microbes. I just want to know is the gene more expressed in different time points. Because there are a lot of microbes in soil, I can not use any reference gene in qRT-PCR, so I was planning to use an external reference, for example DNA. How does this work?

Thank you a lot for the tips!

#2 BioMiha

BioMiha

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 235 posts
9
Neutral

Posted 13 April 2011 - 06:14 AM

You cannot use an external reference DNA because reference genes are used to normalize the qPCR data due to variances in cell number. For example if in one point you take a lot of microbes your qRT-PCR data will tell you there is a lot of your gene in soil. If in another time point you sample much fewer microbes, you will probably also get lower values for your gene. That does not necessarily mean your gene is not expressed in that time point but this is due to a difference in sampling. That's why reference genes are used to normalize sample to sample variations. I would advise you to search for a sequence of a common nucleic acid (e.g. rRNA) that is more or less constantly expressed in soil.




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

©1999-2013 Protocol Online, All rights reserved.