Hello.
I have an exercise to complete where I have to compute the expression of a specific gene.
Two files with results of a real time PCR given to me.
The first one has the results with primers for actin beta and the second one with primers for the specific gene.
The samples in each run was 10 (with the wild type) and each sample repeated 3 times.
Which you think is the best way to calculate the gene expression ?
Thank you.
Hello,
to analyze real time PCR expression you will make a subtraction of expression"level" between a gene of interest and a "housekeeping gene" also called reference gene. This calculation is known as a delta-Ct value (the expression level is called Ct in most thermal cycler softwares). When you want to compare the expression of a gene in various conditions (treatment/control, kinetic,etc.) the 2-deltaCt is the most widely used method.
For details, please check the Livak and Schmittgen publication that describe this calculation: http://dingo.ucsf.edu/twiki/pub/Cores/BioinformaticsCore/QPCR/Livak_Schmittgen2001.pdf
regards
Hi Use Livak et al, they have outlined different ways to do it.
http://dingo.ucsf.edu/twiki/pub/Cores/BioinformaticsCore/QPCR/Livak_Schmittgen2001.pdf
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