The basic principles of good primer design are the same between endpoint PCR and qRT-PCR, but there are a few things to keep in mind. For SYBR green qPCR, your amplicon size should ideally be between 100-150nt and you really don't want to go above about 250nt. Also, the overall efficiency and specificity of the PCR reaction needs to be very good. For endpoint PCR, we can often overlook things like poor amplication efficiency or formation of a low level of primer dimer. For qRT-PCR with SYBR green, any dsDNA that forms in your reaction will contribute to the signal, so it's very important that no primer dimer forms (and you should check your primers for predicted self-dimer and hetero-dimer formation before ordering them.) Also, if you are using the delta-delta-cT method, a core mathematical assumption is that the amplification efficiency of your reference gene primer set and your gene of interest are the same, so primer efficiency needs to be checked and it needs to be very high.

I would also suggest thinking about where you design your primers with respect to intron/exon junctions. Do your genes of interest have isoforms? If so, are you targeting all of them, or just some? Think about this at the design stage and not when the reviewers ask about it! Also, it is good practice to design primers in such a way that any genomic DNA which ends up in your RNA preparation either cannot be amplified at all, or results in off-target signal to warn you that it's there. If you design your assay so that one primer spans an exon-exon junction (eg, half the primer is in exon 4 and half the primer is in exon 5), then your primer shouldn't anneal to gDNA and you won't get any amplification if gDNA is present. When this isn't possible, you can design primers in consecutive exons (eg, forward primer is in exon 4 and reverse primer is in exon 5). This way, if gDNA is present, you will get a large product that warns you that gDNA contamination is present (this will show up on a melt curve and on an agarose gel) so you can clean up your RNA and repeat the qPCR. The junction spanning design in particular is a pain to do well by hand. I like using Primer-blast for this - it works great if you keep nearly all the default settings and just change the amplicon size to be appropriate for qRT-PCR.

Hello, Bilal Ahmad how are you doing today?

No, primers for conventional PCR or qRT-PCR are not the same. The difference will be the size of the amplified fragment. For conventional PRC you can amplified small fragments 100bp to 10kbp. The polymerase has high efficiency to do that. In qRT-PCR we amplified only small fragments 70bp to 150bp, this size is usually enough to detected expression qRT-PCR analysis. Another difference between them, is conventional PCR we are analyzing genomic DNA, so we do not worry about introns and exons in the amplified fragment. In qRT-PCR, we analyze mRNA, so we need to design primers to amplified only exons, avoiding introns and non-coding regions in the gene sequence.

qPCR analysis using SYBR green 150bp fragments should be enough for you. You can setup this in the PRIMER Blast tool from NCBI, and also set design primers in exon regions or not. Blast the design primers with the exon sequence from your target gene to check the specificity. Be careful with different isoforms of your target gene and check whether the design primer will not amplified them.

My best regards… good luck.

Thanks Alot Laura Leighton and Tulio Trio Yoshi aha for the brief explanation