Science Buddies: "Ask an Expert"

Hi,

I am a high school student working on a project using quantitative PCR. 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.

Hi,

I apologize for the delay in answering your question about error bars. I am definitely not an expert on qPCR, but I believe that the error bars, which represent 1 standard deviation from the mean, would require samples run in duplicate. However, can you post an example of your data, showing results of a series of dilutions, perhaps we can help.

Donna Hardy

Hi,

Mrs. Hardy is definitely correct in saying that error bars will require you to do replicates. While the series of dilutions is nice to have, you can do a curve fit on it, but that will only give you the expected value at a given dilution, not an error bar. But seeing the data would help.

Hope this helps!
Aaron Lin