Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum > Real-Time PCR and Quantitative PCR Forum
Register Search Today's Posts Mark Forums Read

Real-Time PCR and Quantitative PCR Forum Real-Time PCR and Quantitative PCR Forum


d(c)t pfaffl method issue!

d(c)t pfaffl method issue! - Real-Time PCR and Quantitative PCR Forum

d(c)t pfaffl method issue! - Real-Time PCR and Quantitative PCR Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 04-19-2013, 11:46 AM
Pipette Filler
Points: 12, Level: 1 Points: 12, Level: 1 Points: 12, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2013
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default d(c)t pfaffl method issue!



Hello all,

I am new to the forum and posting because I can't seem to find anyone in my lab which does work on qPCR.

I am interested in getting some values of expression level for one target gene. All I want to do is to get a d(ct) value which will represent the level of expression for all my 400 samples so I can see how the expression differs over time and space bz doing some mixed effect model.

I have one target gene an three reference genes, the efficiency of the genes vary by more than 10% so I thought about using the pfaffl method to calculate the d(ct) value. I used the following equation : dct= efficiency target ^ct taget / eff ref ^ dct ref.
However, I realized quickly that there is an issue with my results.
Samples which have the highest expression value (lowest ct value before normalization) ends up with the highest d(ct) values and therefore when I try to plot the values for the manuscript, the lowest expressed samples seem to be the highest.

I hope you follow me, it is a bit hard to explain, all I want to say is that my dct values are kinda reversed and the plot does not make sense and is much harder to explain than it should.

Do you know if I made a mistake or if there is smtg I can do to make my results and graph better?

Thanks a lot everybody
Reply With Quote
Reply


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
[Neurofly] The Heisenberg Special Issue of J Neurogenetics Neurofly 2008 Drosophila Forum 0 02-20-2009 09:23 AM
Yeast Digest, Vol 45, Issue 2 Shaoyu Wang Yeast Forum 0 02-05-2009 07:09 PM
Proteins Digest, Vol 22, Issue 15 Dan Guire Protein Forum 0 03-31-2007 10:13 PM
Methods Digest, Vol 17, Issue 23 Sayyari, Mohammad Protocols and Methods Forum 0 10-25-2006 09:11 AM
Scientific method (was Re: Fusion In Utah (again)) Bill Vajk Chemistry Forum 12 09-29-2003 08:30 PM


All times are GMT. The time now is 05:10 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11548 seconds with 16 queries