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
- - - - -

Strange amplification curve in RT-qPCR

amplification curve RT-qPCR

  • Please log in to reply
No replies to this topic

#1 marek82313

marek82313

    member

  • Active Members
  • Pip
  • 28 posts
0
Neutral

Posted 21 March 2014 - 09:00 AM

Dear all,

 

we are setting up a new protocol for a RT-qPCR, for this purpose we are looking for the best control gene for our experiments.

The curves for the control genes are very good, but those for the transcript to be measured are strange.

 

We are trying to use primer pairs for 3 control genes:

- U6

- PRKCZ

- CTCF

 

The template is a cDNA.

Starting from the same sample of RNA, we synthesized the cDNA using 3 primer mixes:

 

Mix A - gene-R + CTCF-R

Mix B - gene-R + PRKC-R

Mix C - gene-R + U6-R

 

the primer "gene-R" contains a tag, meaning that we have about 20 nucleotides complementary to the gene transcript and about other 20 nts that are unique and can anneal to another oligo whose sequence is not present on the genomic DNA ("tag").

 

The qPCR mixes are prepared like these:

Control genes: 

Mix A - CTCF-F + CTCF-R

Mix B - PRKC-F + PRKC-R

Mix C - U6-F + U6-R

 

Gene in analysis

Mix A - gene-F + tag

Mix B - gene-F + tag

Mix C - gene-F + tag

 

While the curves for all the control genes and that for "Mix C - gene-F + tag" are very good (not shown), those for Mix A and B look strange (shown in the attached picture), especially Mix A shows some sort of "bump".

 

What does it mean? Does anybody already experienced Real times with this strange shape? Aspecifics? Dimers? How do I get rid of it?

 

Thanks

 

Mark

 

Curve.jpg

 







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

©1999-2013 Protocol Online, All rights reserved.