Key Steps at the Start Simplify Optimization and Validation
qPCR—like all PCR methods—amplifies the nucleic acids, but qPCR also monitors the process in real time. When putting qPCR to work, scientists must design and validate assays. This poses a variety of challenges in various aspects of this process.
“The key challenges in designing a qPCR assay center on the selection of primer and probe sequences and reporter dyes, particularly in multiplex assays,” says Rod Pennington, a senior research scientist in the Promega (Madison, WI) research and development group. “Also, the choice of oligo sequences and detection chemistries compatible with one’s available real-time qPCR instrumentation can be a difficult balancing act.” He adds, “The more targets one wishes to detect in a multiplex assay, the more precarious the balancing act can be.”
Additional challenges further complicate the design stage. For example, Karen Li, senior product manager of genetic analysis at Thermo Fisher Scientific (Waltham, MA), points out the need to get the most current sequence information and avoid “sequence variation, including SNPs, insertions, deletions, and low-complexity sequences.” With that information, says Li, a scientist then needs to make sure the “assay targets a unique sequence and will not target multiple spots in the genome.”
Features of the target affect how well the PCR works. “When looking at transcripts, it is best to design assays across [the] exon-exon junction so that you are not detecting target genomic DNA,” Li explains. “Consider whether or not you need to detect a specific transcript or if you would like to detect transcript variants of the same gene.” She adds, “For transgenic experiments, make sure you are looking at species-specific targets.”
Details of design
Some advances in qPCR technology make it easier to design new assays. “qPCR instrumentation with multiple detection channels can, to a degree, simplify assay design by expanding options in designing oligos and probes,” Pennington says. “On the reagent side, new probe chemistries continue to be introduced and add flexibility to assay design.”
Scientists can also rely on existing qPCR assays. As Li says, “We have a broad and growing selection of predesigned TaqMan assays that span a range of applications—gene expression, SNP detection, copy number analysis, et cetera—that are designed using extensive bioinformatics analysis that uses the most current sequence information from public databases and performs in silico quality control on assay design to address the challenges.”
For a new qPCR assay, consider using a design tool. As Li points out, there are “a number of assay design tools that help with custom assay design.” She adds, “Our Custom Assay Design Tool—www.thermofisher.com/cadt—features a ‘custom plus’ pipeline option, which will perform the same bioinformatics analysis that is done on our predesigned assays.” For instance, this tool “typically designs assays with a smaller amplicon for better efficiency in the PCR reaction and provides options for transgenic experiments and for transcriptspecific targets,” Li says.
Optimizing the assay
Once a scientist designs an assay, there’s more work to do, such as optimizing it. “The success of a qPCR assay is dependent upon a large number of variables [that] must be optimized, such as component concentrations and thermal cycling parameters,” Pennington explains. “The optimization matrix for a qPCR assay can be very complex and should include testing of representative sample material.”
The level of optimization needed depends on the quality of the design. “Good assay design is critical to minimizing the work needed to optimize a qPCR assay,” Li says. “Optimization can be challenging if you are not mindful of designing primers, and probes, at uniform temperatures.” If the design is not constructed carefully enough, more work is required to make the assay run as efficiently as possible. As Li explains, “If you don’t do the up-front work of optimizing your assay design, you will need to optimize your PCR reaction to find the best annealing/ extension temperatures that work not only for your primers and probes but also for your master mix and instrument.”
The optimization can be more difficult in some situations than in others. “If sample material is hard to come by, availability of the material for assay optimization experiments may be problematic,” says Pennington.
For the best outcomes in optimization, scientists might seek specific features in qPCR technology. “Instrumentation with high-throughput potential can aid in optimization,” Pennington says. “High-throughput potential may come in the form of fast thermal cycling, easy interfacing with robotic pipetting stations, or other features.” Running multiple thermal cycling profiles at the same time also speeds up how fast a scientist can optimize an assay.
The reagents also impact optimization. “New master mix products are introduced fairly often and in some cases allow the removal of certain parameters, such as MgCl2 or enzyme concentration, from the optimization matrix,” Pennington explains. “This can reduce the scope of experiments needed to get the assay up and going.”
Validation is needed
After going through the steps to design and optimize a qPCR assay, it needs to be validated—to show that it really does what it is supposed to do. “From my perspective, the most challenging part [of] qPCR assay validation is the assessment of variance introduced by different qPCR platforms,” says Mark Laible, a molecular diagnostics scientist at BioNTech Diagnostics (Mainz, Germany). “It can be quite frustrating to notice that the instrument type you developed the assay on is prone to large inter-instrument variations.”
To reduce the odds of such problems, he says, it is best to “select an instrument with low inter-instrument variation early on during technical development.” To make such a selection, says Laible, one should “run the assay on at least three different instruments of a specific model.” He adds, “Most instrument manufacturers are helpful in putting you in touch with other labs [that] have such an instrument.”
The required effort in validation often depends on the application at hand. In many cases, commercially available assays will do the job. When needed, however, completely novel qPCR assays can be developed for specific applications. By combining the right qPCR and reagents, plus experience and design tools where needed, scientists can design, optimize, and validate assays.