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Genetic Engineering & Biotechnology News | GENengnews.com | AUGUST 2014 | 23 a method, but the technology also comple- ments sequencing. Real-time PCR is a vital step in the sequencing reaction used to vali- date sequencing results or for target verifca- tion prior to sequencing. Innovation drivers include lowering re- action volume and increasing throughput, without sacrifcing accuracy. Regardless of market industry, research scientists continue to demand faster and inexpensive real-time PCR instruments and lower reagent costs. Integrated with robotics and liquid han- dlers, Roche's LightCycler ® 1536 system runs 1,536 samples in a reduced reaction volume of 0.5–2 μL, in approximately an hour, great- ly reducing reaction volume and cost. "Master mixes are fairly optimized right off the bat; they may require a tweak here or there, but optimization is no longer a major issue. The challenge for the customer is always price. Reducing the cost of the individual reac- tion by reducing the required volume is key," comments Joe Donnenhoffer, Ph.D., lead tech- nical service scientist. "Additionally, tempera- ture homogeneity across the plate is a chal- lenge. Often outer and inner wells will get dif- ferent results due to temperature fuctuation; temperature homogeneity across the plate allows use of all wells with accurate results." "Instrument calibration is another often overlooked laboratory chore that should be automated. When users have to periodically calibrate an instrument, it decreases their confdence in the consistency of the results," continues Dr. Donnenhoffer. "The trend is toward instruments that require no calibra- tion, enabling users to generate consistent results run after run." In the diagnostic world, PCR is more of a black box providing a yes or no answer. In the research feld, users want to know quantity— "how much?" is a harder question to answer. Training has expanded over time as re- search scientists incorporate more and more technologies into their toolbox, and dedicat- ed PCR experts are not typically present in all laboratories. Basic and advanced training will teach users what is going on in the reac- tion, how to interpret the results, and how to determine their next steps. Intellectual Property Concerns According to Caroline Tsou, global mar- keting director for molecular and synthetic biology at Agilent, intellectual property and the associated licensing costs continue to weigh heavily on the market and constrain broader development and use of quantitative PCR. As some of that IP comes off patent in 2016 and beyond, opportunities for ad- ditional development are anticipated, espe- cially in the diagnostics space. Though more advanced capabilities have been developed such as microarrays or NGS, there is still a use for faster, cheaper tools for quick diag- nostics or validation. OMICS The Roche LightCycler® 1536 instrument is a plate-based thermal block cycler with integrated real-time, online detection capabilities. When integrated with robotics and liquid handlers, the instrument can run 1,536 samples in a reduced reaction volume of 0.5–2 μL in approximately an hour. Absolute quantifcation by digital PCR is made possible by instruments that run thousands of individual reactions in parallel. One such instrument, the QuantStudio™ 3D System from Life Technologies, uses a sealed chip technology to streamline workfow. The instrument can generate up to 20,000 data points and enable more controlled sample loading and less reaction dropout than droplet-based approaches. the added power of three lasers Results: Simplifed eight-color immunophenotyping of whole blood without compensation Immunophenotyping of whole blood samples to determine count and percentage of lymphocyte subsets has become increasingly important in immunology research. The guava easyCyte™ 12 system makes this level of multiplexing simple and economical by providing absolute count information from low volume samples. In the example shown, we performed an eight-color assay using whole blood staining in a simple, no-wash format to clearly and easily identify B cells, NK cells, CD4 and CD8 T cells as well as naïve and memory subsets of these populations. The comprehensive analysis provided key subset identifcation while utilizing no compensation. Figure 2. 10 µL of adult blood was stained with a cocktail of antibodies and lysed with guava® lysing solution. Samples were then acquired on the guava easyCyte™ 12HT system. Lymphocyte cells (CD45+) were gated into a SSc vs. CD3 plot. T cells (CD3+ and CD45+) were gated into a plot comparing CD4 and CD8 positive cells. CD4 and CD8 naïve and memory cells were further discriminated by separating CD45RA (Naïve T cells) vs. CD62L (memory T cells) in each population. To separate the natural killer (NK) and B cells from the lymphocytes, CD3-negative cells were gated into a plot comparing CD19 (B cells) and CD16+56 (NK cells). Separation is visible and is comparable to previously published data. Conclusion The guava easyCyte™ 12 system offers expanded detection capability and analytical power with the addition of the violet laser to the existing blue and red lasers. The added parameter selection provides increased fexibility in designing a variety of assays. Applications ranging from multiplexed cell health analysis to multicolor immunophenotyping of whole blood subsets bring more power to the hands of researchers. Coupling plate-based analysis on the guava easyCyte™ microcapillary system with EMD Millipore's fow cytometry reagents and kits provides the ideal platform for a multiplex approach to cellular analysis. Explore what's possible. Visit: www.emdmillipore.com/guava CD45(+) Side Scatter CD45 PerCP-Cy5.5 Side Scatter CD3 Brilliant Violet™ 421 Non T Lymphocytes T Cells CD3(-) CD3(+) CD19 Brilliant Violet™ 510 CD16/CD56 PE CD4 PE-Cy7 CD8 APC-eFluor® 750 CD8(+) CD4(+) B Cells CD4+ T Cells CD8+ T Cells NK Cells Central Memory Cells 31.1% 17.3% Effector Memory Naïve Cells 45.4% 5.2% Terminally Differentiated CD62L FITC CD45RA APC Central Memory Cells 16.2% 36.1% Effector Memory Naïve Cells 27.9% 19.8% Terminally Differentiated CD45RA APC CD62L FITC Lymphocytes Population Count (cells/µL) Lymphocytes 1298± 16 T Cells 967 ± 19 CD4 T Cells 632± 21 CD8 T Cells 234± 8 B Cells 63± 5 NK Cells 217± 3