Pushing the Limits of PCR: Considerations for Assay Development in High-Throughput qPCR and Digital PCR Platforms

Part 1: Robust Assay Development for Multiplex qPCR and SNP Genotyping in Miniaturized Volumes on a High-throughput Instrument.

Quantitative real-time PCR (qPCR) is one of the most valuable tools in biological research and is utilized in a wide range of applications including detection of Single Nucleotide Polymorphisms (SNPs), nucleic acid quantification, and gene expression analysis. Advancements in genomic sequencing technologies have expanded the overall amount of genetic information available, subsequently creating a need for laboratory instrumentation and reagents that enable accurate and cost-effective PCR analysis for routine genetic studies.

BHQplus® probes from LGC Biosearch Technologies are compatible with many commercially available master mixes and can be used in real-time and endpoint PCR platforms, including the Array Tape® Platform. A popular choice for PCR-based SNP analysis, BHQplus probes are industry-leading in quality, specificity, and economic value. The IntelliQube® is the latest evolution of the Array Tape technology, integrating liquid handling with qPCR analysis in miniaturized reaction volumes. The variety of fluorophores available for BHQplus probes align well with IntelliQube’s five fluorescence channels, providing users the added capability of multiplexing SNP genotyping assays.

Reverse transcription-qPCR (RT-qPCR) is widely used for gene expression analysis, RNA virus detection, or RNA quantification experiments. RT-qPCR can be performed directly from RNA in a one-step process, or using a two-step process where RNA is first converted to cDNA and analyzed by PCR in a subsequent reaction. In both cases, BHQ® probes and the IntelliQube offer a powerful combination for generating RT-qPCR data enabling reproducibility, sensitivity, and flexibility in terms of fluorophore choice and multiplexing capability.

This webinar will discuss the use of BHQ and BHQplus probes in combination with the IntelliQube for gene expression analysis and SNP genotyping. Using samples from a variety of research markets, we will demonstrate successful SNP genotyping in a multiplexed format utilizing four fluorescent probes. We will also assess the use of BHQ probes for gene expression analysis, briefly discussing experimental design considerations while highlighting multiplexing capabilities. Combined with the use of BHQ and BHQplus probes and the automated workflow of Array Tape, the IntelliQube proves to be a versatile and powerful platform for a variety of PCR applications.

Part 2: Digital PCR as a Tool to Support the Molecular Diagnosis of Infectious Diseases

Jim Huggett will
briefly introduce digital PCR (dPCR) and then discuss the application of the methods to infectious disease analysis. The talk will describe sources of error and explore the methods reproducibility and will use a number of examples from Jim’s group at LGC, which forms part of the UK’s Designated National Measurement Institute for Bioanalytical measurement, to demonstrate the potential of this powerful technique. Technical problems will also be highlighted and the potential role for dPCR in the clinical analysis of infectious diseases such as measuring drug resistance or to support established methods like qPCR will also be discussed.

Speakers

Luke Linz, Ph.D., Laboratory Operations Manager, LGC Douglas Scientific

Luke Linz is the Laboratory Operations Manager at LGC Genomics in Alexandria, MN. He is responsible for overall operation and administration of the laboratory with specific focus given to customer needs, research requirements, assay development, quality control and work flow processes. Linz joined LGC Genomics in 2013 after receiving his Ph.D. from Iowa State University where he conducted research in the field of microbiology.

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Jim Huggett, Ph.D., Principal Scientist, Nucleic Acid Research, LGC, and Senior Lecturer, Analytical Microbiology, University of Surrey

Jim Huggett, Ph.D., Principal Scientist, Nucleic Acid Research, LGC, and Senior Lecturer, Analytical Microbiology, University of Surrey
Jim Huggett is a Principal Scientist at LGC (formerly the Laboratory of the Government Chemist and the UK’s designated National Measurement Institute for chemical and bioanalytical measurement) and a Senior Lecturer at the University of Surrey. He received his Ph.D. at Cardiff University studying mechanically regulated genes in bone remodelling. He then spent seven years as a Senior Research Fellow at University College London (UCL) investigating molecular diagnosis of infectious diseases and developing methodology and capacity for application in a developing world setting. Now he conducts measurement research into molecular, genomics and nucleic acid analysis applicable to infectious diseases and other fields such as cancer diagnosis and stratification. Much of his research is currently focused on characterisation of high accuracy measurement as well as strengthening the traceability of measurements to understand sources of error and improve reproducibility. Dr. Huggett is also an honorary Lecturer at UCL and is Managing Editor for the Elsevier journal Biomolecular Detection and Quantification.

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