Dear Garima,

Short answer: It is right if you do reverse transcriptase PCR and work with cDNA.

It is not right if you work with genes that contain introns and you do qPCR with genomic DNA.

Long answer: The answer to your question depends on what you want to look at in the PCR. If you want to do RT-PCR and work with cDNA and want to know if your gene of interest is expressed and in what amount it makes sense to use the coding sequence.

Keep in mind though what the coding sequence means  - this region equals the open reading frame and does not include introns and 3´and 5´ untranslated regions. In many cases it makes sense to design primers for RT-PCR by using the whole hypothetical cDNA sequence including the untranslated regions. I preferably put my primers in the untranslated regions but thats up to each researcher him/herself.

If you do PCR with genomic DNA and know/are not sure if your gene contains introns your template for primer design should be of course genomic DNA and not the coding sequence. The coding sequence is essentially a sequence of introns only. If your primer happens to span an exon-intron border it won´t bind properly and the PCR will not run smoothly.

Thank you friends for your valuable answers.

Yes, I want to do the expression analysis and will be doing Reverse-Transcriptase PCR to carry q-PCR with the cDNA. I will take care of all the parameters.

Is cost an issue? If not then I would go for hydrolysis probes as primer dimer issues are not a problem for your quantification. I guess the next issue is suppliers. In the UK we have it easy suppliers like IDT (www.idtdna.com)  have tools on their website to aid design for common organisms. I have used them many times for hydrolysis probes and the cost is roughly £70 compared to say £13 for standard primers. But the quality of data you get is much better. I appreciate that shipping costs etc. to some overseas countries becomes prohibitive.

Did you use EST from the web for designing primers. If you use a cDNA sequence for primer designing make sure that the primer is designed overlapping the splice junction or an exon -exon junction. This will help you to mitigate the problem of DNA contamination in your cDNA sample using for qPCR.

one more thing to add is that if you are using oligodT For synthesizing your cDNA fromthe mRNA then you can design primers flanking the 3' end but its always safe that you dont design primers at the 5' untranslated area becoz the efficiency of RT will not be 100% and you may not get the full length cDNA during your RT reaction