I did three technical replications for real-time PCR to analyze comparatively 2 copies of the same ancestral gene (A and B, with very specific primers for each one of them) in four different organs cDNA (leaf x stem x roots x flowers). I used Actin as a housekeeping gene. I know that if I calculate Delta CT with Actin (“Gene A - Actin” or “Gene B - Actin”), I can calculate the relative quantification (RQ) and detect at the end the increase or decrease of gene A or gene B expression in other organs compared with the one that I choose. This is part of I want to know with the experiment and I think the resulting values are exactly what I was expecting.
But if I calculate Delta CT by (GeneB CT - GeneA CT), will I have a relative quantification at the end saying the expression of B compared with A, for each organ?
You can not compare (d)Ct-values from different genes. You will need external standards to make the results for different genes comparable.
Another possibility would be to amplify both versions in the same PCR with the same primer pair (if the sequences allow doing this) and to differentiate the sequences after PCR, by sequence specific probes or a restriction analysis. You would at least the information which gene is expressed at a higher or lower level, but if you want to quantify the difference wou aigain will need an external standard (see "quantitative competitive PCR" for more details).
Hi,
For such type of analysis I would suggest not to g for 'relative quantification' but do 'absolute quantification'
Ajay
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