If you have the exon sequence, then you have the cDNA. You'll want to know the intron locations to design qPCR primers.

You should talk to colleagues and read some resources about qPCR experiments BEFORE you do anything else. You need to know how you will analyze your data, whether you are doing a relative vs quantitative experiment, how many biological vs technical samples, etc.

If you start by just ordering some primers without really knowing what you are doing, you are going to waste a lot of time and resources.

As my advisor would say "You can't rush quality". Good luck!

Hi Katie

Thanks for your reply. Actually, we have predicted a genomic region using FGENESH and we found an exon in this genomic region. We exported the exon sequence and need to validate the expression of this genomic region. So, can we design a primer for that genomic region using the exon sequence that we predict

Hi Gragor

the best way would be to have the entire cDNA to allow the design of almost one of the primers across one splice site at most 3' of the gene.

go to the UCSC database and do a blast (in the tools) to get access to the gene/region/informtions (cDNA).

all the best

fred

Hi Frideric

i do not have the entire cDNA this is a predicted genomic region. The bioinformatics tools classified this genomic region as a predicted Exon. Therefore, I was able to extract the sequence of this exon and I think this genomic region is a part of the unknown gene. Therefore, I would like to confirm this with gene expression by designing a primer for gene expression. From the discussion with Kaite, I believe that she is correct and logical since all exons of genes are transcript to RNA then the Reverse transcriptase will convert this RNA to DNA (which is the original exon)

Keep in mind your prediction might not be strand-specific. If you ran the prediction on DNA data, maybe some sort of assembly, the exon/gene prediction is unstranded. This means you don't know if the exon/gene is coded on the positive or negative DNA strand - you just know it's in this region. You might have to design your primers both ways to capture it.

OK, given the context of the research, you are going to want to start by using regular PCR to see if the region of the gDNA can be amplified at all.

In-silico predictions often have errors. You'll save yourself a LOT of stress if you can show that the genomic region exists in the DNA.

Besides, you'll need to clone that region of the genome to make a standard curve for your qPCR. It's a "win win" to check for the gDNA first.