Early real-time PCR was focused on perfecting instrumentation. Instrumentation is no longer a rate-limiting step in the production of quality data; instead, usability, software, and analysis are at the forefront.
“The big problem with real-time PCR is not the amount or complexity of data, but whether or not data are any good. Everyone can get traces, especially with poorly designed primers and probes,” explains Sam Ropp, Ph.D., senior business unit marketing manager, GXD Consumables, Bio-Rad Laboratories. “The focus has shifted from hardware improvements to tools that impact data quality. When users get better data, they can have confidence in the first run.”
A set of guidelines, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE), seeks to address a challenge with the widespread adoption of quantitative PCR, the lack of a universal standard to substantiate the quality of data.
Bio-Rad provides transcriptome-wide content for the human and mouse genomes—an assay for every protein-coding gene. (The company is close to providing similar content for the rat genome.) For over 20,000 assays per transcriptome, researchers receive validation data based on the MIQE Guidelines.
Assays are also prevalidated and arranged into subsets of biologically relevant information, leading to predesigned assay plates that correlate to biological signaling pathways and disease states, along with the ability to customize the plates to an individual’s liking. Run files supplied with plates allow users to import gene information and validation data into the instrument software, automating set-up and analysis.
Controls help determine if data reflect a biological event or an experimental artifact. Reference genes and controls are built into predesigned assay plates to check sample quality, reverse transcription, presence of PCR inhibitors, and genomic contamination.
“Every technique and application evolves over time; real-time PCR is no different,” continues Dr. Ropp. At present, real-time PCR is acquiring process improvements “from sample isolation to interpretation of results.”
The goal is to incorporate flexibility and ease of use so that users receive answers quickly and feel confident that their results are bulletproof. “It gets down to data quality and the experimental workflow—filling in the gaps to make [real-time PCR] easier, quicker, more efficient, and price effective,” concludes Dr. Ropp. “That is the future.”