The enhancement of PCR with real-time quantitative methods takes PCR from a workhorse laboratory preparatory method for molecular biology to an extremely powerful diagnostic and analysis tool for clinical and research applications. Studies of gene expression are some of the most common applications of qPCR. It is also increasingly used for clinical diagnostic testing. The technique is sensitive enough to detect DNA or RNA from an infectious disease, or even early stages of cancer.
Practical applications of real-time PCR, however, are plagued with problems and complications that slow down data collection or decrease the quality of results. These problems include contamination, amplification bias, poor efficiency and sensitivity, and unsatisfactory throughput or cost. CHI’s “Quantitative PCR” meeting held recently in San Diego brought the scientific community together to share strategies and solutions for making the best use of qPCR.
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Although technological improvements can enhance a qPCR experiment greatly, some of the most effective fixes are straight forward and easy to apply with any system. Marwan Alsarraj, product manager for Bio-Rad Laboratories’, gene expression division, taught a three-hour workshop at the meeting on optimizing qPCR assays. The course covered optimization of the assay and troubleshooting common problems. “By spending more time designing and qualifying a good primer set, the researcher saves time and prevents many pitfalls down the road.”
According to Alsarraj, running a standard curve by serial-fold dilution of template DNA with SYBR Green or Eva Green fluorescent dye can help provide valuable insight into the robustness of an assay, yielding important information such as the PCR efficiency, dynamic range, reproducibility, sensitivity, and specificity of the assay. This experimental approach could be used to ensure a properly optimized PCR assay.
This type of validation assay has helped many scientists improve the efficiency of their qPCR experiments. “I remember a scientist with a primer set generating a low PCR efficiency. By utilizing the melt curve, I checked the specificity of the reaction to make sure there were no primer dimers. We set-up a serial-fold dilution of the template DNA. It was clear by the number of peaks present that more than one product was amplified as the amount of input nucleic acid was dramatically decreased. When we got rid of this primer set and redesigned the primers, the PCR efficiency jumped from 60 to 95 percent.”