Hi Luigi,

I hope I got your question correctly. However, on which strand of gene should have no consequences for amplification (on which strand your gene is located only tells you in which direction the gene is transcribed). Nevertheless, both strands are part of the gene and no matter what the direction of transcription is, your primers will always amplify both strands. Usually, you call your primer which have the same 5' to 3' sequence as the coding strand the fw primer and the primer with the the same 5' to 3' sequence as the template strand rev primer - but this is independent from the direction in which the gene is transcribed.

The same is true when you design your primer for your mRNA quantification. The direction of transcription on the chromosomal level is irrelevant.

When you do mRNA quantification via PCR you first have to do a reverse transcription to created a single-stranded cDNA. Your primer which has the same 5'-3' sequence as the mRNA (except for a T insteads of an U) will be your fw primer and the primer which has the same 5'-3' as the cDNA will be you reverse primer - however no matter how you name the primer - they will still amplify your sequence.

If you design your primers for mRNA quantification you should search for the mRNA sequence for your gene (e.b. NCBI nucleotides) and use this sequence for designing your primers ( as I mentioned already above, the location or the direction of transcription on a chromosomal level is completly irrelevant for this, so don't worry about this). This is important as this squence is most likely not the same sequence as the genomic sequence, due to the exon-intron structure of the gene. However, if you want to to a quantification of your mRNA you might search for primers which are spanning an intron, because if the intron is big enough you will avoid amplification due to contamination of genomic DNA.

I hope this helps answering your question

If you want to look at gene expression (mRNA), you'd use a Reverse Transcription reaction with a polyT primer to create double-stranded cDNA from your mRNA.

The cDNA is a "DNA version" of the mature mRNA transcript (5'UTR, exons 3'UTR, polyA tail).

You need to know something about how your gene is annotated (e.g. exons vs introns; start codon) before you can design any qPCR primers.

for a fragment of DNA, 5'-3' , forward primer is any 15-25 bases as is in start, and reverse primer is the last 15-25 bases, just that for reverse primer you have to convert it as a reverse complement. Reverse it and complementary of it. Thats it. simple and clear. ty.

Thank you! I overlooked the RT step; of course, measuring the cDNA won't affect the assay.