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Although he sees himself as a technology evangelist, Brant’s career spans academic research, the biotech industry, and several years as a paleontologist hunting dinosaurs in the Australian outback.
After 40 peer-review publications and two US Patents (for DNA silver staining and DNA fingerprinting) he left academia to introduce and evangelise real-time PCR technology in Australia and New Zealand for Applied Biosystems way back in 1997, a role he is reprising now with NanoString Technologies. While working at Corbett Research he helped pioneer and introduce HRM (high resolution melt) technology (an acronym you can blame on him!). Over the years Brant has had many roles including Senior Research Officer, Product Manager, Global Marketing Manager, and even Chief of Operations at Australian Age of Dinosaurs. Academic Career: Brant graduated with a PhD and First Class Honors from the Australian National University (Canberra) in 1988. His first PostDoc was at the Racheff Chair of Excellence in Plant Molecular Genetics in Knoxville Tennessee USA from 1988–1993 He then moved back to Australia as Senior Research Officer at the CRC for Tropical Plant Pathology, University of Queensland, from 1993-1995 |
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Brant will be speaking on:
RT (Reverse Transcription): The Elephant in the PCR Room
The study and interpretation of gene expression (GX) signatures is a powerful approach to investigate complex, responsive systems biology. Quantitative real-time RT-PCR (RT-qPCR) is one of the most important spanners in our PCR toolbox and considered by many to be a stalwart of molecular diagnostics. However, the implementation of GX signatures is notoriously finiky, with very few signatures graduating to widespread use. The blame for this is often directed at the samples themselves (the biology), or blamed on the technical skills of individual technicians. In fact, the real answer stems from fundamental issues with the reverse transcription (RT) step. Unfortunately, RT is an unavoidable step for any PCR-driven GX analysis: It is used to enzymatically convert mRNAs in the original sample into a representative pool of synthetic, single-stranded, complimentary DNA (ss cDNA) molecules. It’s this ss cDNA pool that is compatible with the PCR (the original mRNA molecules are not). However, while poor quantitative reliability of the RT step has long been recognised, technical innovation and genuine progress has been at a standstill for decades. The only proffered solution has been seemingly endless (and impractical) rounds of optimisation of replicate RT reactions, employing several different RT enzyme variants to empirically assess quantitative linearity and reproducibilty for a set of representative target mRNAs. Such optimisations are to be repeated for every system studied. Validation of this sort is so onerous and expensive it’s little wonder it is rarely done.
In recent years an entirely new technology, nanoString, has emerged that avoids the need for an RT step entirely. This new approach directy counts and quantifies individual mRNA molecules from a sample with digital precision. It uses no enzymes, no sample splitting, and requires only a single hybridisation event to assess all target mRNAs and controls in a single-tube assay. Armed with this complete, orthogonal validation method, we can at last objectivly investigate the issues that frustrate RT-qPCR. This talk will present simple, real-world experimental data that identifies the cause and extent of irreproducibility in RT-qPCR and how to assess it and address it in your experimental plans.
RT (Reverse Transcription): The Elephant in the PCR Room
The study and interpretation of gene expression (GX) signatures is a powerful approach to investigate complex, responsive systems biology. Quantitative real-time RT-PCR (RT-qPCR) is one of the most important spanners in our PCR toolbox and considered by many to be a stalwart of molecular diagnostics. However, the implementation of GX signatures is notoriously finiky, with very few signatures graduating to widespread use. The blame for this is often directed at the samples themselves (the biology), or blamed on the technical skills of individual technicians. In fact, the real answer stems from fundamental issues with the reverse transcription (RT) step. Unfortunately, RT is an unavoidable step for any PCR-driven GX analysis: It is used to enzymatically convert mRNAs in the original sample into a representative pool of synthetic, single-stranded, complimentary DNA (ss cDNA) molecules. It’s this ss cDNA pool that is compatible with the PCR (the original mRNA molecules are not). However, while poor quantitative reliability of the RT step has long been recognised, technical innovation and genuine progress has been at a standstill for decades. The only proffered solution has been seemingly endless (and impractical) rounds of optimisation of replicate RT reactions, employing several different RT enzyme variants to empirically assess quantitative linearity and reproducibilty for a set of representative target mRNAs. Such optimisations are to be repeated for every system studied. Validation of this sort is so onerous and expensive it’s little wonder it is rarely done.
In recent years an entirely new technology, nanoString, has emerged that avoids the need for an RT step entirely. This new approach directy counts and quantifies individual mRNA molecules from a sample with digital precision. It uses no enzymes, no sample splitting, and requires only a single hybridisation event to assess all target mRNAs and controls in a single-tube assay. Armed with this complete, orthogonal validation method, we can at last objectivly investigate the issues that frustrate RT-qPCR. This talk will present simple, real-world experimental data that identifies the cause and extent of irreproducibility in RT-qPCR and how to assess it and address it in your experimental plans.
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"2018 NGS, dPCR & qPCR Symposium" is managed by Gene Target Solutions. Ph: (02) 8090 2675. info@genetargetsolutions.com