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Aug 1, 2014 (Vol. 34, No. 14)

Real-Time PCR: Yet More Worlds to Conquer

  • The polymerase chain reaction (PCR), invented about three decades ago, soon entered mainstream use thanks to an ongoing series of refinements.

    One particularly important refinement, introduced about two decades ago, is the “real time” quantification of DNA. The idea is to trace the rising level of DNA throughout the amplification step, and not just measure the final amount of amplified product. This idea turns standard PCR into real-time PCR, or quantitative PCR.

    Real-time PCR has become the most widely used nucleic acid detection technology. It is routinely used in academic research, in applied testing settings such as food-safety or veterinary testing, and in molecular diagnostics. It continues to replace many older detection methods due to simple readouts, high sensitivity, and multiplex and quantification capabilities, as well as ease of use, cost effectiveness, and throughput flexibility with only moderate equipment investments.

  • Click Image To Enlarge +
    Qiagen’s QuantiNova kits have a built-in tracking system for visual identification of correct pipetting.

    According to Peter Urbitsch, Ph.D., head of the global assay technologies business at Qiagen, real-time PCR technology has evolved and diversified in multiple directions. Available formats include tubes, microarrays (96-, 384-, and 1,536-well plates), and capillary and rotor variants. Detection principles include SYBR green and probe-based detection, with the latter being increasingly diversified into FRET, Scorpions, TaqMan, and others. In addition, multiplex detection formats are being developed using different dyes and quencher molecules.

    Innovations are driven by dissemination into new application areas and user profiles. Essentially, the technology is becoming easier to use and accessible to novices while providing more complex information faster and at lower costs. This development can be compared to computer technologies, which evolved from bulky “specialist equipment” to powerful and convenient end-user devices.

    As real-time PCR continues to replace traditional technologies, less experienced users become routine users. While some instruments are small, simple, and accessible to almost anyone and deployable at nearly any bench, other platforms are targeting high-throughput data generation and require robust chemistry that allows automated handling and reaction set-up at room temperature.

    Procedures include a higher degree of process controls (such as target controls via multiplexing) or operational controls (such as the visual pipetting control in Qiagen’s QuantiNova kits). This is particularly important in complex applications such as multiplex detection of various targets and integration or cross-linking of multiple scientific questions such as genotyping, mRNA and miRNA profiling, or copy number variation (CNV) analysis.

    A common bottleneck for both real-time PCR and other technologies such as next-generation sequencing (NGS) is the consolidation and interpretation of data and results, increasingly requiring bioinformatics tools that support the interpretation of the biological meaning of gene expression data, such as Qiagen’s Ingenuity Pathway Analysis and Ingenuity iReport.

  • Beyond Instrumentation

    Early real-time PCR was focused on perfecting instrumentation. Instrumentation is no longer a rate-limiting step in the production of quality data; instead, usability, software, and analysis are at the forefront.

    “The big problem with real-time PCR is not the amount or complexity of data, but whether or not data are any good. Everyone can get traces, especially with poorly designed primers and probes,” explains Sam Ropp, Ph.D., senior business unit marketing manager, GXD Consumables, Bio-Rad Laboratories. “The focus has shifted from hardware improvements to tools that impact data quality. When users get better data, they can have confidence in the first run.”

    A set of guidelines, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE), seeks to address a challenge with the widespread adoption of quantitative PCR, the lack of a universal standard to substantiate the quality of data.

    Bio-Rad provides transcriptome-wide content for the human and mouse genomes—an assay for every protein-coding gene. (The company is close to providing similar content for the rat genome.) For over 20,000 assays per transcriptome, researchers receive validation data based on the MIQE Guidelines.

    Assays are also prevalidated and arranged into subsets of biologically relevant information, leading to predesigned assay plates that correlate to biological signaling pathways and disease states, along with the ability to customize the plates to an individual’s liking. Run files supplied with plates allow users to import gene information and validation data into the instrument software, automating set-up and analysis.

    Controls help determine if data reflect a biological event or an experimental artifact. Reference genes and controls are built into predesigned assay plates to check sample quality, reverse transcription, presence of PCR inhibitors, and genomic contamination.

    “Every technique and application evolves over time; real-time PCR is no different,” continues Dr. Ropp. At present, real-time PCR is acquiring process improvements “from sample isolation to interpretation of results.”

    The goal is to incorporate flexibility and ease of use so that users receive answers quickly and feel confident that their results are bulletproof. “It gets down to data quality and the experimental workflow—filling in the gaps to make [real-time PCR] easier, quicker, more efficient, and price effective,” concludes Dr. Ropp. “That is the future.”

  • Changing Industry Segments

    In terms of adoption of the technology, the core academic segment is mature. Translational and applied market segments are growing as new applications are being enabled.

    “Technology miniaturization means smaller components and instruments that are less expensive, smaller, lighter, and easier to deploy in the field. Integration from sample prep to answer, multiplexing to get more dimensions of information with extremely high specificity and sensitivity at a low cost point and efficient workflow, is making the technology attractive for emerging market segments to replace more laborious and time-consuming existing detection modalities,” remarks Chris Linthwaite, vice president, genetic, medical and applied sciences, for Thermo Fisher Scientific.

    An example is microbiological applications where real-time PCR is replacing cell culture. Technicians used to have to plate, let the colonies grow, pick homologous colonies, and then type them. Real-time PCR allows detection with very few copies, reducing a once lengthy workflow to a few hours.

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