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
- - - - -

Act1 mRNA decay is not linear

qPCR act1 mRNA decay

  • Please log in to reply
No replies to this topic

#1 aznbabie

aznbabie

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 18 September 2014 - 08:05 AM

Hello everyone,

 

I am calculating mRNA half lives between two genes that I am interested in. I performed an analysis of the housekeeping gene (Act1) on a Quantity (no of copies) vs Time (min). The graph is not linear (it's polynomial), making the results for my other genes invalid.

 

Though the other two genes that I was looking at had "regular" looking decays.

 

What kind of troubleshooting should I do?

 

My general protocol:

-Preformed RNA Polymerase shutoff with thiolutin (3ug/mL) over 60 mins (8 timepoints)

-Extracted RNA with a kit

-Performed cDNA first strand synthesis and cDNA cleanup with a kit

-qPCR different dilutions of a pooled sample of all timepoints for "actual" standard on the actual experimental plate

-Found the best dilution (1:5), performed cleanup of qPCR 1:5 dilutions

-Followed qPCR map and performed 1:5 dilution for all timepoint samples and ran plate

 







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

©1999-2013 Protocol Online, All rights reserved.