delta delta CT method - (Jul/14/2008 )
i'm using delta delta CT method to calculate the fold change in gene expression between two stages. I'm using housekeeping genes for normalization. When i calculate the fold change relative to normalized gene, sometimes i get less than 1 (log10 value in negatives). i realized that this is due to the differences in expression levels of housekeeping and target genes. can someone suggest me how to deal with this problem? what does the negative log means?
Am not sure that I completely understand. Could you give a hint about your calculation? Actually, where do you use the logarythm?
An excellent text on deltadelta Ct could be reached via Applied Biosystems site.
I would also use the efficiency corrected version of the delta delta Ct method which is described by roche in their realtime pcr manuals... is much more precise than without like AB does... although a little more complicated... but in the end only a bit of mathematics-....
the only problem (or danger) with the efficiency corrected method is that you can introduce more error than with ddCt if the efficiency is not accurately measured. so you need good standard curves over a broad dilution range.