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OMICS RT-PCR: Ready for Prime Time? Kate Marusina, Ph.D. The conceptual and practical simplicity of quantitative real-time PCR (qPCR) has made it a choice tool for many molecular analysis applications. Because of its speed, sensitivity, and specifcity, qPCR has been displacing conventional PCR in the vast majority of its applications. Some believe that it is only a matter of time before qPCR becomes a major player in diagnostics. A large swath of publications reporting reverse transcriptase qPCR (RT-qPCR) data underscores a great interest in this technique. Yet there still exists a relative lack of consensus in how best to perform the RT-qPCR experiments. "We can't simply assume that RT-qPCR always accurately quantitates gene expression levels," says Chaminda Salgado, head of CMC bioassay and genomics at NDA Analytics. "Each step of the procedure has technical challenges. Optimization and validation of each step is key." He adds that the "advancement of qPCR into the diagnostic realm will depend on standardization of at least four key technical components: Sample handling and assessment; RT strategy—enzyme selection and RT priming; normalization during analysis; and increase in hardware speed—for pointof-care applications." To date, standardization has been ham- pered by lack of suffcient experimental details in scientifc reports. Frequently, qPCR publications omit critical analysis parameters and justifcation for reference gene selection, hindering critical evaluation of the quality of the results. To enable inter-laboratory comparisons, qPCR-based clinical diagnostic assays will require more stringent standardization. Salgado emphasizes that primer design, reverse transcriptase selection, and choice of reference genes are vital underpinnings for the progression of RT-qPCR from a research technique to a market-ready in vitro diagnostic tool. The frst step toward this goal may be implementation of the Minimum Information for Publication of Quantitative RealTime PCR Experiments (MIQE) guidelines. These call for reporting of minimal essential and desired information to ensure qPCR relevance, accuracy, correct interpretation, and repeatability. Properly implemented, the MIQE guidelines could help promote consistency among laboratories and potentially zero in on the methods that show potential utility for diagnostic applications. Researchers at the Dana-Farber Cancer Institute have developed an alternative enrichment method that does not involve enzymatic amplifcation. DISSECT avoids enzymatic steps as it is entirely based on repeated cycles of denaturation and hybridization on magnetic beads coated with wildtype target gene sequences. "QPCR is a sensitive method, but many clinically important analyses require detection of minority events below qPCR current limits of sensitivity. Quantitation of rare mutations on the background of wild-type DNA is of prime importance in several felds of medicine," says Mike Makrigiorgos, Ph.D., from the Dana-Farber Cancer Institute. "Specifcally, subclonal mutations in cancer cells may determine if a particular tumor is resistant to chemotherapy. We envision that monitoring the quantity of these mutations during cancer treatment may inform management decisions for the remainder of the therapy or subsequent rounds of therapy." Dr. Makrigiorgos' team focuses on development of techniques for identifcation of rare events and translating them into diagnostic applications. A few years ago, they developed COLD-PCR (co-amplifcation at lower denaturation temperature) to enrich samples with Fluorescence signatures generated from the combination of four On Probes and four Of Probes distinguish a penicillin-resistant variant (green) of the bacterial TEM gene from two variants (blue, red) with extended-spectrum antibiotic resistance. Brandeis University mutations irrespective of where they occur in DNA sequence. The technology takes advantage of a small but detectable difference in melting temperatures of mismatched sequences. Denaturing and re-annealing genomic DNA creates mismatched DNA duplexes. NEWS Genomics & Proteomics > Evotec, Dow AgroSciences Team Up in Chemical Proteomics Effort Evotec and Dow AgroSciences are collaborating in an efort to support compounds in development at the latter frm. Under the terms of the research agreement, Dow will leverage Evotec's chemical proteomics services. Evotec will be performing quantitative chemical proteomics services to clear up Dow's phenotypic screening results. Financial terms were not disclosed. > CLC Bio, SciEngines Pair Sequence Analysis Software with Massively Parallel Computing As part of an ongoing partnership, CLC Bio and SciEngines are integrating the former frm's sequence analysis software with the newest model of the latter's 24 | FPGA-based RIVYERA computers. The frms said that, rather than using heuristics, through their partnership it's now possible to fnd exact matches between sequences at a speed of 11.5 trillion cell updates per second per server. > Rutgers University Biorepository Wins $44.5M from NIMH to Support Genomic Research RUCDR Infnite Biologics, the Rutgers University-based biorepository, has won a Cooperative Agreement award worth $44.5 million from the National Institute of Mental Health (NIMH) to support the NIMH Center for Collaborative Genomics Research on Mental Disorders through the collection and processing of blood and tissue samples from NIMH-funded scientists nationwide. With this funding, August 2013 | GENengnews.com | Genetic Engineering & Biotechnology News RUCDR Infnite Biologics will also provide statistical and analytical consultation for such collaborative projects. > Genomatica Licenses DNA Design Software from TeselaGen Genomatica and TeselaGen have inked a multiyear agreement though which the former frm is licensing the latter's software, which uses synthetic biology approaches to build and modify DNA. Additionally, the companies are working together to enhance TeselaGen's software in eforts to meet Genomatica's objectives. > Hitachi High-Tech, Base4 Working to Develop NanoporeBased Sequencing System Hitachi High-Technologies and Base4 Innovation have entered into a collabora- tive technology development agreement to develop a long-read, single-molecule, nanopore-based DNA sequencing system. The frms said the system will be based on technology developed by Base4 and instrumentation developed by Hitachi High-Tech. > Through Partnership, NYGC Buys BioNano Sequencing System The New York Genome Center (NYGC) and BioNano Genomics have entered into a strategic partnership through which the nonproft organization is purchasing an Irys™ System from the sequencing frm. The partnership provides the NYGC with access to a long-read sequencing system. For BioNano, it presents the opportunity to place a "commercial-ready technology" in a largescale research setting, the company said. n