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
- - - - -

RT-PCR standard curve dilutions


  • Please log in to reply
2 replies to this topic

#1 pcrmossad

pcrmossad

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 19 April 2010 - 01:29 PM

Hi all,

I am using RT-PCR for validating microarray data for some time now. usually I use 1, 0.1, and 0.01 dilutions to generate my standard curve. in rare cases I have used 0.2 .

my question is should I always use these dilutions? cant I use 1, 0.5, 0.25 etc dilutions to generate a standard curve? what are the disadvantages of using this kind of dilutions apart from wasting control samples?

thanks in advance.

#2 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,198 posts
109
Excellent

Posted 20 April 2010 - 04:39 AM

You should always have standards that span the area where your samples are. So if you except samples to be maximum 10 fold downregulated, you don't need to do 0.01 dilution in your standard curve.
Usualy more wider dilutions are used because they span all possible concentrations, but if diferent dilutions have different amplification efficiency (due to inhibition for example) it may skew the slope of the standard curve. In that case for concentrations around 0.5 - 0.25 the standard curve with 0.5 and 0.25 dilutions could be more accurate. It really depends on the range of your samples.

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 pcrmossad

pcrmossad

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 20 April 2010 - 12:50 PM

You should always have standards that span the area where your samples are. So if you except samples to be maximum 10 fold downregulated, you don't need to do 0.01 dilution in your standard curve.
Usualy more wider dilutions are used because they span all possible concentrations, but if diferent dilutions have different amplification efficiency (due to inhibition for example) it may skew the slope of the standard curve. In that case for concentrations around 0.5 - 0.25 the standard curve with 0.5 and 0.25 dilutions could be more accurate. It really depends on the range of your samples.


thanks again !




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

©1999-2013 Protocol Online, All rights reserved.