In Search of Better Real-Time PCR
Real-time quantitative PCR (qPCR) has become the industry standard for the detection and quantification of nucleic acids. However, the lack of consensus among researchers on how to best perform and interpret qPCR experiments is a major hurdle for advancing the technology. This problem is exacerbated when insufficient experimental detail is given in published work, impeding the ability of others to accurately evaluate or replicate reported results.
In extreme instances, incongruous pre-assay conditions, poor assay design, and subjective data analysis methods have led to the publication of irrelevant or misleading data. A 2002 paper on measles, mumps, and rubella (MMR) provided evidence that supported a link between the MMR vaccine and autism. Later examination established that the original conclusions were based on flawed real-time PCR data; subsequent publications demonstrated no plausible link between the vaccine and autism.
To promote the integrity of submissions to peer-reviewed journals, consistency between laboratories and experimental transparency, researchers established Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards. MIQE’s goal is to develop a consistent approach to qPCR data reporting that encourages detailed auditing of experimental processes, analysis, and results whereby the quality of the work and reliability of the conclusion can be evaluated.
Bio-Rad Laboratories Inc. offers scientists technology become MIQE compliant including a MIQE qPCR iPad application or app, available for free through Apple’s App Store. The app provides immediate access to MIQE quidelines, and uses color-coded checklists to distinguish between essential and desirable MIQE requirements. Researchers can send their project checklists to the MIQE database where the information will be used to further refine the guidelines.
For RT-qPCR experiments, quality control of the RNA starting material is an essential checkpoint addressed by the MIQE guidelines. These require that RNA integrity be measured by gel electrophoresis at the very least, but preferably by microfluidics-based RNA analysis using a system such as the Bio-Rad Experion.
Bio-Rad has paired its CFX96 Touch and CFX384 Touch real-time PCR systems with qbasePLUS software from Biogazelle NV, Zwijnaarde, Belgium, helping eliminate erroneous data, remove sample-specific non-biologic variation, and perform inter-run calibrations to reduce technical variation between samples. By enhancing the reliability of the qPCR data, qbasePLUS conforms to MIQE. Both the CFX96 and CFX384 systems can direct data export to qbasePLUS software or to MIQE-recommended RDML file formats. Researchers can handle both small and large experiments as well as combine data from different experiments using qbasePLUS software as the repository database.