I always understood fold change calculations following a qPCR to be calculated by 2^-(ddCT). Is this incorrect, my mentor thinks it is? Potentially they are thinking of something else. Could you please include literature mentioning the correct way to calculate fold change.
I ran a co-culture experiment with HeLA cells and bacterial members common of the vaginal microbiota. I used siRNA to knockdown LAMP3 antiviral gene.
Hi,
There are indeed multiple ways of calculating fold change from qPCR data with absolute (using a standard curve) and relative (using the relative expression of a normalising gene to account for sample variation, as in the ddCT method) being the two major groups. I have attached two fairly comprehensive reviews that should help explain the differences between methods and why you would one over another. However typically ddCT is the most popular approach.
http://www.biotechniques.com/multimedia/archive/00011/BTN_A_05391RV01_O_11901a.pdf
http://strategy.gene-quantification.info/
Hope this helps
Hi,
There are indeed multiple ways of calculating fold change from qPCR data with absolute (using a standard curve) and relative (using the relative expression of a normalising gene to account for sample variation, as in the ddCT method) being the two major groups. I have attached two fairly comprehensive reviews that should help explain the differences between methods and why you would one over another. However typically ddCT is the most popular approach.
http://www.biotechniques.com/multimedia/archive/00011/BTN_A_05391RV01_O_11901a.pdf
http://strategy.gene-quantification.info/
Hope this helps
Hi
The delta-delta C2 method is not incorrect, however the assumptions needed for this calculation should be first carefully analyzed first. If some of these are not proper, appropriate parameters should be taken into account before concluding the fold up and down regulation.
If you are looking for another method we are calculating the relative genes exprssion using Pfaffl formula. It takes into account also reactions efficienty:
Pfaffl MW. A new mathematical model for relative quantification in real –time RT-PCR. Nucleic Acids Res. 2001; 29: 2002-2007.
Search Pubmed for MIQE, the guidelines for qPCR analysis; e.g.
https://www.ncbi.nlm.nih.gov/pubmed/19246619
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