qPCR error bars - confused! - (Nov/26/2014 )

Hi,

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!  

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

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!