Hints for Real-Time PCR (qPCR) Assay Design

It’s a common question Thermo Fisher Scientific gets from people who’ve done a lot of gel-based PCR over the years and designed hundreds of primer sets, but never for real-time PCR. You might be asking yourself “Do I need to do anything special?” First off, size definitely matters when it comes to amplicons in real-time PCR. Be sure not to make your products too large, as this can contribute to a less-than-optimal PCR efficiency. Instead, try to keep them somewhere between 50 and 150 bases long.
Second — and this is critical for quantitative experiments — don’t design oligos to a mismatch or to a known polymorphic site, as this will cause inefficient binding during the first few PCR cycles and shift Cts to the right. Instead, always confirm that you have the correct sequence. Third — be careful about designing to sequences that aren’t unique. Be especially mindful of homologues, which often differ very little, if at all, in certain regions, and will therefore cause erroneous signal.

Try to always design at least one oligo to a region that’s unique enough so that your assay won’t give unwanted signal. Finally, you never want to design oligos to low-complexity sequence, including repetitive regions that certainly won’t be unique in your genome or transcriptome.
There are lots of ways to get assays that fulfil all the criteria I mentioned. One way, of course, is to do all the bioinformatics work yourself on the context sequence, then use a program such as Primer Express Software to do the assay design. You then just order individual oligos. One of the really nice things about Primer Express Software is that it designs every set of oligos to work at Universal Cycling Conditions, meaning you never have to optimize real-time PCR conditions. Speaking of convenience, you should know about 2 other handy options for getting high-quality TaqMan Assays: by either having Thermo Fisher Scientific design them for you through one of their Custom services, or by purchasing a pre-designed Assay. In either case, you can spare yourself the trouble of doing bioinformatics, your assays will all work at Universal Conditions, and they’ll amplify with 100% efficiency— all of which should make you very happy. If you want to try out a pre-developed TaqMan® Assay for gene expression, we have excellent coverage for lots and lots of species. We even have pre-developed TaqMan Assays for other applications, including SNP genotyping, microRNA and other small RNAs, copy number assays, and lots more.
And don’t worry — if we don’t have your assay already, we can always custom design it for you.