When it comes to real-time PCR in drug discovery, Richard Kurtz believes that MIQE guidelines will help create a clear path to better results.
“Quality data is paramount for the successful development and potential approval of a drug candidate”
Polymerase chain reaction (PCR) has evolved into a readily automated, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the industry standard for the detection and quantification of nucleic acids for multiple applications, and particularly for the quantification of mRNA expression levels.
However, a lack of consensus among researchers on how to best perform and interpret qPCR experiments presents a major hurdle for advancement of the technology. This problem is exacerbated by insufficient experimental details in published work, which impedes 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. An early report published in 1998 in The Lancet has been cited as evidence supporting a link between the Measles, Mumps and Rubella (MMR) vaccine and the development of autism. Later examination established the original conclusions of the paper were based on flawed real-time PCR experiment data and the interpretation has been formally retracted. Subsequent publications have convincingly demonstrated there is no plausible link between the vaccine and autism, but the debate and doubt continues outside of the scientific community and MMR vaccination rates have dropped in several countries. Unfortunately such a retraction of conclusions based on qPCR data is not an isolated incidence and there are multiple examples in the literature where data and conclusions presented either cannot be independently evaluated for quality or are obviously flawed due to inappropriate analyses or experimental design. When such data is used as a basis for future experimental design, including identification of potential therapeutic candidates, the consequences can be dire.
Accurate, repeatable, and reliable qPCR testing can help companies avoid pursuing the wrong compound or target. Providing sufficiently detailed reports of experimental design in early stage drug discovery research can potentially save millions of dollars by avoiding wasted working hours or reagents. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards can help companies and researchers avoid these drug development pitfalls.
The goal of the MIQE guidelines is to structure the current approach to qPCR data reporting into a consistent format that encourages detailed auditing of experimental processes, analysis, and results whereby the technical quality of the work and reliability of the conclusions can be evaluated. The guidelines thus promote the careful examination of qPCR results to enhance integrity of submissions to peer-reviewed journals, consistency between laboratories, and experimental transparency. Adoption of and compliance with these guidelines will help researchers more accurately report data and replicate experiments, ultimately saving time and money. Implementing these guidelines is imperative in continuing the growth of qPCR into an accurate and reliable nucleic acid quantification technology.
When it comes to providing scientists with the tools to be MIQE-compliant, Bio-Rad Laboratories, Inc. is paving the way. Recently, Bio-Rad entered into an arrangement with Biogazelle to exclusively distribute the qbasePLUS data analysis software with its CFX96TM and CFX384TM real-time PCR detection systems, a program that enables users to annotate their experiments with MIQE-compliant experimental details. The qbasePLUS software in combination with these real-time PCR systems provides customers with improved accuracy in their qPCR experiments and speeds up standardized data analysis.
qbasePLUS software allows for the rapid examination, identification, and elimination of erroneous data, together with normalization to remove sample-specific non-biologic variation and inter-run calibration, which can remove the technical variation between samples analyzed in different runs. By enhancing the reliability of the qPCR data, qbasePLUS assists researchers in conforming to the MIQE guidelines.
Using the CFX96 and CFX384 real-time detection systems with qbasePLUS software accelerates research through automated, fast calculations and direct import of data from the system to the software. This combined solution provides researchers with the flexibility to handle both small and large experiments as well as combine data from different experiments, using qbasePLUS software as the repository database with correct error propagation and tracking.
A critical step in a PCR reaction is the annealing of the primers to their target sequences. It has to be performed at the right temperature for the primers to anneal efficiently to their targets, while preventing nonspecific annealing and primer-dimer formation. The thermal gradient available on all of Bio-Rad's real-time PCR detection systems, is a valuable tool for determining the optimal annealing temperature of primers and probes to ensure specific and robust amplification of amplicons.
Upstream in the real-time qPCR workflow, Bio-Rad's ExperionTM automated electrophoresis system provides rapid, objective, and reproducible assessment of RNA sample integrity, an essential step in the MIQE guidelines. The Experion system is documented in the original MIQE publication as ideal for the collection of information about the RNA sample quality. In addition to generating a virtual gel, an electropherogram, and calculating the 28S/18S ratio, the Experion system's software calculates and reports an RNA quality indicator (RQI) value that reflects the integrity of the RNA sample. This value allows the researcher to rapidly select RNA samples that meet the integrity criteria required for an accurate quantitation of the sample.
As the industry continues to recognize the importance of the MIQE guidelines it will become increasingly necessary to ensure that laboratories and corporations are following them. Simplifying compliance facilitates not only drug discovery and development, but basic biological research. Investing in a qPCR system and failing to follow guidelines that will ensure that quality data is being produced may result in a wasted investment.
It can be anticipated that additional vendors will soon offer solutions to assist researchers in maintaining MIQE compliance. Top instrument makers and reagent vendors are supporting the idea to expand MIQE awareness and compliance. Bio-Rad has trained sales and support staff to help researchers comply with the guidelines. They share this training through educational meetings focused on using the MIQE guidelines to generate quality qPCR data.
Quality data is paramount for the successful development and potential approval of a drug candidate. Following MIQE standards will help strengthen the quality qPCR data needed to move a candidate forward and decreases the chances of focusing on the wrong target or having a drug candidate fail.
Richard Kurtz, PhD is senior marketing manager at Bio-Rad Laboratories. Prior to joining Bio-Rad, Kurtz served as field applications manager for MJ Research Incorporated. Kurtz earned his PhD from Northwestern University and a BA in Molecular Biology from Colgate University.