Hi Ramon,

your way to analyze seems to be the best way, but be sure that your designed primers will be sufficiently specific to each gene. if your genes have enough exons, I would choose different exons junctions to distinguish all genes (for each gene and therefore each primers pair, one primer on the exon junction, one in the exon, Tm and characteristics at maximum ressemblance, and all amplicons around 120-150pB length).

fred

I don't thinks that's the correct way. The dCt values are sequence-specific, and comparing dCt values is reliable only within the same sequence, not between different sequences. However, if a semi-quantitative analysis of rather huge expression differences is sufficient, that might do anyway. A precise and reliable quantification to compare different sequences will require using external standards.

Ramon:

If these 9 genes from the same gene family are very similar in sequence, then your major problem will be designing primers that are highly specific for each gene. This problem may be compounded by the possibility that the genes are expressed at very different levels. So, for example, you might have particular trouble detecting specific expression of one of the genes with low expression if there is another gene family member with higher expression that is weakly amplified by the primers that you designed for the low-expressing gene. Such amplification could give you the wrong impression about the expression of the lower expressing gene. (Designing primer pairs with PCR products of different melt temperatures could help with diagnosing such an issue.) One possible place to look to design highly specific primers would be in the 3' (or possibly 5') UTRs of the 9 different mRNAs. Since these regions may be less conserved, you might find sequences there that are closer to being unique.

An example of using 3'-UTR sequence to develop quantitative PCR primers for 3 very similar genes is here:

Article Quantitative Tissue and Gene-Specific Differences and Develo...