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qPCR error bars - confused!

qRT-PCR error bars qPCR statistics

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#1 toffee89



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Posted 26 November 2014 - 06:27 AM



I need help with some qRT-PCR error bar calculations. I have carried out all of my qPCR with my gene of interest and of my housekeeping gene and have calculated my fold-changes for my gene of interest with the delta-delta-Ct calculation. However putting error bars on my graph showing the fold-changes is confusing me. What data should I be using to calculate this? I can't use the normalized Ct values as this is not correct for fold-changes and when I convert my Ct values (for my calibrator and experimental) using the delta-delta-Ct calculation and then get the SE for that my error bars are bigger than my fold-change. Can anybody shed some light on this and where I am going wrong? I can't use the software that comes with the qPCR machine as it is getting confused with how my plates are arranged.  


Thank you in advance!  

#2 Trof


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Posted 19 December 2014 - 03:17 PM

Just curious, what software comes with your machine (i.e. what is your machine) that gets confused by plate arrangement?

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#3 miRman



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Posted 26 November 2015 - 05:55 AM

Hi, have you read Livak & Schmittgen 2001? Changing  the exponential process (PCR amplification) into linear comparison (fold change),as it happens in the method  (2^-delta-delta-Ct),need to be also applied for the upper and lower values of the error. You need to calculate the standard deviation of your delta-Ct values and calculate the positive and negative error values from delta-delta-Ct +SD and delta-delta-Ct -SD using the same 2^ conversion. If this is not done, especially in the case when the "treated" group is lower than 1 (control) simple SD error bars may cross the X-axis. The correct error bars for this type of data are always different size in + and - direction.


Hope this helps!

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