MIQE Guidelines for RT qPCR Experiments
Blog Jan 13, 2014
Scientists and journals have been slow to adopt the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines that were established in 2009 to bolster the reliability of real-time PCR (qPCR) and reverse transcription qPCR (RT-qPCR) data. To help boost adoption, Bio-Rad scientists published a brief and practical guide that concisely summarizes the key steps required to produce high-quality, reproducible data for labs conducting RT-qPCR experiments.
We spoke co-author of the guide, Bio-Rad Field Application Specialist, Sean Taylor, to understand more the importance of MIQE, the low adoption rates and what the future may hold.
AB: Why is MIQE so important?
Sean Taylor (ST): MIQE provides a framework that standardizes the way all labs perform qPCR experiments. This ensures high quality and reproducible data within and between labs. Before the guidelines, researchers were conducting qPCR experiments primarily based on teachings from senior scientists or students who have learned from previous labs without any critical examination of those teachings. This has resulted in a wide variability of the techniques and methodologies employed at each step in a qPCR experiment. These steps include experimental design; sample handling and extraction; RNA isolation, purification and qualification; reverse transcription;, and the qPCR reaction using validated primers and reference genes. Mistakes made at any of these steps can and has resulted in artifactual results and article retractions. Since the scientific community relies on the published, peer-reviewed literature to design future projects, research can be dramatically impaired if the papers used for guidance are in fact misleading. Adhering to MIQE is important for ensuring that data published with qPCR permits the steady progress of research in the many fields that use this technique.
AB: Why have rates of adoption been so low?
ST: MIQE has not been more widely adopted due to the belief within the many labs that have been using the technique for several years that they are already conducting experiments correctly. In practice, science is based on the design of experiments and strict adherence to protocols that have been passed down from one generation of researchers to the next. Therefore, changing the way we perform a particular experiment that has been conducted the same way for years is like changing a lifestyle or moving to a foreign country. Even with good justification to change, there is always the fear that the resulting data may be different and may no longer support the conclusions of previous work.
AB: How does your paper encourage researchers to adopt MIQE?
ST: This article concisely contrasts both good and poor practice at each step of a qPCR experiment. Scientists will be able to easily relate to the examples cited in the two "Do's" and "Don'ts" of qPCR tables, which we hope will be used as a quick reference to qPCR experimental design and conformance to the MIQE guidelines. We also hope that the brevity and practicality of the article will encourage the scientific community to read and adopt the steps described for high quality, reproducible data.
AB: How are publications without MIQE currently received? Is there scepticism regarding the reliability of the data?
ST: This entirely depends on the journal and the reviewers. An increasing number of journals are standing behind MIQE, an acknowledgement that much of the data published with qPCR is difficult or impossible to reproduce. These journals therefore require the submission of supplemental data showing conformance of experiments to the key criteria listed in the JMMB article.
However, there remains a large proportion of the scientific community and journals who have ignored MIQE mostly for the reasons previously stated, so there is still much work to be done to nudge the scientific community in the direction of the guidelines.
We remain highly optimistic that MIQE will continue to be adopted and will grow as continued awareness is generated. Within Bio-Rad, our Field Application Scientists educate all labs using our qPCR instrument and reagent technologies about the benefits of MIQE and many of these labs have changed their practices to conform. We have found that there is skepticism regarding the reliability of published qPCR data particularly after a lab has undergone training and at least some of the "poor" practices described in the JMMB article are shared by many labs which leaves them asking "Why has no one ever taught me this?" to which the answer is "Because you were taught by someone who was not taught."
AB: Do you envisage a time when MIQE will be a requirement for publication?
ST: We certainly hope this would be the case and I suspect this opinion is shared with the thousands of scientists who have read and referenced MIQE in their papers. Scientists’ only currency is their published articles, which translate to grants that sustains the cycle of research. The whole process falls apart if the data published do not reflect the biology tested, which results in misleading conclusions that confounds and slows the engine of research. Scientists want to produce high quality data that stand the test of time in the literature on which their peers can build. We believe that by continuing to generate awareness for MIQE the research community will embrace the guidelines for the good of all.
Sean Taylor provides more information in the video Following the MIQE Guidelines for RT qPCR Experiments