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22 | AUGUST 2014 | GENengnews.com | Genetic Engineering & Biotechnology News tion 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 stan- dard to substantiate the quality of data. Bio-Rad provides transcriptome-wide con- tent 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 valida- tion data based on the MIQE Guidelines. Assays are also prevalidated and arranged into subsets of biologically relevant informa- tion, 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 fles 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 refect 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," con- tinues Dr. Ropp. At present, real-time PCR is acquiring process improvements "from sam- ple isolation to interpretation of results." The goal is to incorporate fexibility and ease of use so that users receive answers quickly and feel confdent that their results are bulletproof. "It gets down to data quality and the experimental workfow—flling in the gaps to make [real-time PCR] easier, quicker, more effcient, 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. Transla- tional and applied market segments are grow- ing as new applications are being enabled. "Technology miniaturization means small- er components and instruments that are less expensive, smaller, lighter, and easier to deploy in the feld. Integration from sample prep to answer, multiplexing to get more dimensions of information with extremely high specifcity and sensitivity at a low cost point and effcient workfow, is making the technology attractive for emerging market segments to replace more laborious and time-consuming existing detec- tion modalities," remarks Chris Linthwaite, president of genetic analysis at Life Technolo- gies (now part of Thermo Fisher Scientifc). An example is microbiological applica- tions where real-time PCR is replacing cell culture. Technicians used to have to plate, let the colonies grow, pick homologous colo- nies, and then type them. Real-time PCR al- lows detection with very few copies, reduc- ing a once lengthy workfow to a few hours. The fastest-growing segment of the real-time PCR market is enabling vast levels of multiplex- ing and density, running the same test with dif- ferent samples or interrogating the same sample for different elements. Digital PCR, an absolute quantifcation, expands utility versus cannibal- izing real-time PCR. The Life Technologies QuantStudio 3D Digital PCR System, for ex- ample, is a chip-based platform that generates up to 20,000 data points. In personalized medicine, NGS and quan- titative PCR are complementary tools. NGS platforms answer open-ended questions with lots of unknowns, but typically real-time PCR is the fastest and most cost-effective mo- dality for detecting the same thing over and over again. PCR is translating to the clinic as more CE-IVD-labeled or 510K-cleared in- struments are approved by regulatory bodies. Eventually, compact, personalized, highly de- centralized, and push-button-answer instru- ments will be run bedside in the clinic. The market will continue to mature and enter production environments, integrating with robotics and plate readers and allowing tens of millions of PCR reactions. PCR is a detection tool; value gets added later, in data interpretation. To help users make the most of this stage, industry lead- ers hope to introduce the SaaS (software as a service) model and cloud-based software. Users may be able to access and share data (and update software) more easily. In addi- tion, they may be able to purchase software modules as needed. Innovation Drivers PCR is not only the most sensitive way to do diagnostic testing, replacing ELISA as Real-Time PCR Continued from page 18 OMICS 642 nm 488 nm 405 nm New guava easyCyte™ 12 system increases multiplexing capabilities Multi-laser fow cytometers can dramatically increase the capability of cell analyses. Traditionally, expensive and complicated instrumentation has been required for such comprehensive, multi parametric analysis. By contrast, the popular guava easyCyte™ benchtop fow cytometers have made fow cytometry more accessible and cost-effective for cell biologists in diverse settings. The guava easyCyte™ systems are microcapillary fow cytometers, which do not use sheath fuid, enabling absolute cell counts, low sample volumes and are easier to maintain than traditional fow cytometers. Here, we present data from a new, high- performance system, the guava easyCyte™ 12 instrument, which uses three lasers with ten fuorescent and two scatter detection channels, increasing the amount of information derived from each sample. This system also includes the powerful InCyte™ data acquisition and analysis software, a fexible, intuitive program for answering a variety of research questions for both tube- and plate-based assays. The guava easyCyte™ 12 fow cytometry system offers expanded detection capabilities and analytical power without sacrifcing ease of use. Results: New violet laser provides greater choice and flexibility in designing a variety of assays The guava easyCyte™ 12 instrument has an additional violet laser (compared to the other guava easyCyte™ models) which provides ten fuorescent channels for designing their multiplexed experiments. The performance of each guava easyCyte™ 12 channel was evaluated using whole blood staining with CD4 antibodies conjugated to different fuorochromes in a no-wash assay (Figure 1). The data demonstrate excellent separation and identifcation of CD4 T Cells in every channel. This provides researchers greater choices with regard to the channels and fuorophores they wish to use to design specifc multiplexed experiments. Multicolor flow cytometry on your benchtop: Figure 1. 10 µL of adult blood was stained with directly conjugated anti-human CD4 antibodies for 20 minutes and lysed with guava® lysing solution. Samples were then acquired on the guava easyCyte™ 12HT system. Data were all assessed with SSc vs the CD4 staining for each channel.The CD4+ T cell population was clearly separated from the monocytes, granulocytes and the remaining population in all ten fluorescent channels. easyCyte and InCyte are trademarks and guava, EMD Millipore, and the M logo are registered trademarks of Merck KGaA, Darmstadt, Germany. BS-GEN-14-10237 06/2014 © 2014 EMD Millipore Corporation, Billerica, MA USA. All rights reserved. Brilliant Violet™ 421 (BLU-V-Log) FITC (GRN-B-Log) APC (RED-R-Log) Brilliant Violet™ 510 (GRN-V-Log) PE (YEL-B-Log) APC-eFluor® 750 (NIR-R-Log) Brilliant Violet™ 605 (YEL-V-Log) PE-Cy5.5 (RED-B-Log) Brilliant Violet™ 650 (RED-V-Log) PE-Cy7(NIR-B-Log)