Stephen Bustin, Ph.D., professor of molecular science at Barts and the London School of Medicine’s Institute of Cell and Molecular Science, will present at “qPCR2009.” Dr. Bustin is currently working on a real-time quantitative (qPCR) assay for the detection of Clostridium difficile, the bacterium responsible for a huge number of hospital-acquired infections. He expects that the assay will be more sensitive, more informative, and much less costly than current PCR-based assays.
Such assays, he notes, cost in the $20–$30 range per assay, and dedicated instrumentation is required. His goal is to develop a set of affordable tools that allow not just detection, but also a more detailed molecular characterization of C. difficile. Currently, he notes, there are many problems in the field of qPCR, and the problems are proving to be durable.
Dr. Bustin authored a paper citing such limitations in 2000, “but we haven’t progressed a lot since then,” he observes. In fact, “the field is littered with papers that are meaningless,” with the problems beginning with sample preparation, and extending to “how you normalize, analyze, and report results.”
“It is clear that a high percentage of publications utilizing qPCR technology, and especially those aiming to profile cellular RNA levels, report poorly designed, executed, and interpreted experiments and results,” Dr. Bustin states.
“Considerations of mRNA transcription, in vivo stability, regulation by miRNAs, tissue specificity of splice variants, allele-specific difference in expression, the lack of concordance between most mRNAs and their specified proteins, and the critical importance of post-translational modifications and questions of tissue heterogeneity all describe serious issues that are not being addressed in an adequate manner,” he concludes. “It will require a significant amount of courage, and a sea change in attitude from the research community to deal with this quagmire.”
On a positive note, there are several developments that Dr. Bustin finds encouraging. Among these he cites the introduction of less expensive, optimized reagents that make reaction assembly simpler and more consistent; the development of more intuitive analysis software to help with assay setup and project management; the introduction of advanced algorithms that allow more accurate quantitation; and the extension of the technology into novel areas such as high-throughput, nanoliter qPCR—specifically, microfluidic digital PCR, which is “an exciting new development that extends the scope of qPCR technology.”