Login

Join for Free!
114699 members


Error Bars--QPCR

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Error Bars--QPCR

Postby scienceluvr » Sat Apr 03, 2010 1:13 am

Hi,

I am a student working on a project using quantitative PCR at the Wayne State University School of Medicine. I would greatly appreciate it if anyone could answer a question for me about error bars.

I am trying to add estimated error bars to my graphs. The graphs are fold change in gene expression graphs that were constructed using the efficiency of PCR and delta delta cT values. There are 20 different genes I'm graphing on the X-axis, and the fold changes in expression are on the Y-axis. The gene I used for normalization was ACT, that gave me delta cT values. Then I compared effects of different drugs on the gene expression, for delta delta cT values. Do you know how I can estimate error, without replicate assays? I have done a dilution series along with each PCR run.

Thank you for your time.
scienceluvr
Garter
Garter
 
Posts: 4
Joined: Fri May 08, 2009 12:13 am

Re: Error Bars--QPCR

Postby clevermizo » Sat Apr 03, 2010 3:04 am

Not sure, although I don't see the point of error bars without replicate experiments. Why not just perform replicate experiments?
clevermizo
Garter
Garter
 
Posts: 7
Joined: Thu Jan 28, 2010 3:48 pm

Postby MrMistery » Sat Apr 03, 2010 2:43 pm

there is no way of estimating the error, you have to do the assay multiple times and calculate the standard deviation. It's a way of showing people that it wasn't just that one experiment that went well; doing it multiple times is the whole point
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
User avatar
MrMistery
Inland Taipan
Inland Taipan
 
Posts: 6832
Joined: Thu Mar 03, 2005 10:18 pm
Location: Romania(small and unimportant country)


Re: Error Bars--QPCR

Postby vastgenome » Mon Apr 12, 2010 6:02 am

Hi,

I think a typical qPCR run would consist of at least 3 technical repeats for each unknown sample. Once you get that, the standard deviation can be calculated from these technical repeats, and can be propagated using geometric averaging rule. the error propagated in the first dCt is carried over when doing ddCt, so they are identical. But at the last step when you are estimating the final 2^(-ddCt), you need to calculate the lower and upper bound of 2^(-ddCt) by incorporating the previously calculated linear errors. As a result, the final error bar will be somewhat asymmetric in the 2^(-ddCt) plot.
vastgenome
Garter
Garter
 
Posts: 11
Joined: Mon Apr 05, 2010 2:21 am

Postby JackBean » Mon Apr 12, 2010 9:49 am

the qPCR run does not have to have three replicates. That depends only on your setting ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5665
Joined: Mon Sep 14, 2009 7:12 pm


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests

cron