I am doing a q-PCR for the gene expression level on different organs in common carp. I usually use beta-actin as my housekeeping gene and the delta CT value is around 16-18. In analyzing my data I use relative gene expression using the Livak and Schmittgen method. I read that gene expression level of house keeping gene is organ dependent hence absolute gene expression level is necessary. What are your recommendations and knowledge about absolute gene expression and when should we use this method?
Thank you.
You should monitor the expression of multiple housekeeping genes by qPCR in order to find one whose expression is stable across the different organs you are looking at.
But in the end in depends of what is your question and what you are trying to compare : multiple genes in each organs or one gene across multiple organs?
If you do per organs comparison (gene A vs gene B in organ 1), the variation of expression across organs of the housekeeping gene you use for normalisation is not a problem.
If you do comparisons across organ (organ 1 vs organ 2 for gene A) then it is of concern and then you refer to my first paragraph to find the most stable housekeeping gene to limit bias.
When you compare gene expression, absolute gene expression is rarely of interest and housekeeping genes normalisation must be considered.
Thank you for your answer, I am comparing 1 gene across 12 organs.
Then screen multiple house-keeping genes to find the one that changes the less between your 12 organs.
Be sure to get the real efficiency for each primer couple.
12 organs * 3 technical replicates * 10 house keeping genes is one 384 plate.
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