The evolution of qPCR: Improving accuracy and reproducibility
By: Roche Life Science
Posted: | Lab Life - Real-Time PCR
The first decade
In 1983, while on a drive through Mendocino County in California, Kary Mullis — a researcher with Cetus Corp. — was deep in thought about a problem that had been plaguing him at work: how to find a way to concentrate a single DNA locus in the presence of millions of similar DNA loci. The answer was polymerase chain reaction (PCR), his Nobel Prize-winning discovery that he employed in his research to understand the underlying genetic cause behind sickle cell anemia.
Early PCR experiments were unwieldy and time consuming, at best. The process required manual thermocycling, and replenishing the heat-sensitive DNA polymerase after every cycle. With standard PCR experiments exceeding 20 cycles, that made for an unreasonable amount of manual labor, and lots of lost time spent waiting for each experiment. Nonetheless, it dramatically reduced the time and steps required to replicate large quantities of DNA for life science research, and today it is one of the more commonly used tools used in research labs.
qPCR and the real-time revolution
Just a decade after Mullis’s PCR discovery, Russell Higuchi and colleagues at Roche Molecular Systems and Chiron demonstrated the discovery of real-time or quantitative PCR (qPCR) by connecting a thermal cycler to a spectrofluorometer. With qPCR, minimal amounts of DNA could be replicated rapidly and detected easily. In contrast to traditional PCR, qPCR can ascertain up- or down-regulation of a DNA sequence, or how much of a specific DNA sequence is present in a sample, rather than simply identify its presence or absence.
The speed, sensitivity, and reproducibility of homogeneous assays and the limited risk of carryover contamination with qPCR have made it the gold standard methodology used for determination of DNA copy numbers, quantitating transcription, and other applications.
In the two decades of qPCR, advances have improved sensitivity and reduced variability to make the technique more reliable. But despite the enhancements to the qPCR process, some variability remains in the preparatory steps of sampling and nucleic acid extraction.
Evolving qPCR standards
PCR thermal cycler technology has evolved to include more robust functionality in an ever-decreasing price point, paired with increasingly reliable, less expensive reagents. This has led to ongoing automation of workflows that require less hands-on time, freeing technicians for higher-value tasks.
But as throughput accelerates, more data is available for analysis. The need for consistent quality and data control has become increasingly obvious. Selecting the right instrumentation and reagents can help to set the stage for ensuring accurate and reproducible results.
MIQE guidelines encourage consistency, reproducibility
The widespread application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have established a baseline that increases transparency, promotes global consistency and helps to ensure the integrity of testing outcomes, and therefore the value of the resulting scientific literature.
This attention to consistency and transparency in publication has led to ongoing advances in technology and technique. Advances in instrumentation, reagents, and analysis software has led to improvement in qPCR accuracy and reproducibility, to the benefit of all life science research.