Documenting Real-Time PCR
In February 2009, twelve internationally recognized experts published a long-awaited set of guidelines for real time PCR experiments after more than a decade of public discussion of how to standardize the method and its reporting. These, guidelines called Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE),1 can be found online at www.rdml.org/miqe.php.
The spirit of the guidelines is to standardize and streamline the real-time PCR workflow from the early planning stage through publication. Many real-time PCR experiments currently suffer from a lack of standardization and detail in publication. Some of them are flawed by poor experimental design. Although the guidelines are barely a year old, awareness and use of MIQE is not spreading as quickly as many of the thought leaders had hoped. Instrument and reagent vendors are helping out by providing MIQE compliant products and MIQE training resources for customers.
Sample preparation and RNA integrity
High-quality RNA is fundamental to a successful real-time PCR experiment. Variation in sample sources and preparation can result in significant variations in the outcome of an experiment. MIQE guidelines suggest using the best quality samples possible, and careful assessment of extracted RNA. A measurement of the ratio of optical density at 260 nm and 280 nm can prove that the RNA is devoid of protein and phenol contamination. However, electrophoretic analysis is required to assess the integrity of the RNA itself.
The Experion Automated Electorphoresis system from Bio-Rad Laboratories (Hercules, Calif.) is an example of a technology that is being optimized for MIQE compliance in qPCR. Experion is an automated, microfluidics-based electrophoresis system that combines quantitative and qualitative analysis to provide rapid assessment of RNA sample integrity. “It’s the ideal method for collecting quantitative information about the RNA sample quality as documented in the original guidelines description,” says Rachel Scott, senior product manager Gene Expression Division, Bio-Rad.
Full compliance with MIQE guidelines requires the reporting of the sequence of all oligonucleotide probes. Hundreds of real-time PCR papers have been published that would be very difficult to reproduce due to insufficient experimental detail. However, the exact sequence of many commercial probe and primer pairs are not available, which makes it especially difficult to comply with this particular requirement.
Thermo Fisher Scientific (Waltham, Mass.) is tackling this issue head-on by making all sequence information for their Solaris probes available to users. This allows the customer to map the assay within the target gene. “The MIQE guidelines specify that researchers must provide the sequence information of their probe and primer pair. Unlike other suppliers, we give complete sequence information with each Solaris qPCR Gene Expression Assay, making it easier for customers to publish their work,” says Ben Jackson, PhD, product manager for Thermo Fisher Scientific. “I think we’re alone in doing this. We provide full sequence information.”
The oligonucleotides in the Solaris assay kits include modifications to optimize the assay, but these are not divulged with the sequence information. Only the sequence is required for MIQE-compliant publication.
The company also offers seminars that help customers learn proper notation and other procedures for planning, executing, and publishing MIQE-compliant real-time PCR experiments. “Reading the MIQE paper gives customers a great head-start in understanding the level of detail they need to provide in reporting qPCR experiments,” says Jackson.
Compliant tools for data analysis
Data analysis is perhaps the part of MIQE compliance that requires the most support from vendors. MIQE guidelines mandate a change in some types of terminology, and some methods of data analysis.
An important aspect of correct data analysis is the use of real-time PCR data markup language (RDML). RDML provides a universal standard for reporting and sharing qPCR data and incorporates correct analysis of raw data, assay normalization, and assessment of experimental variability. These changes are straightforward on a mathematical and computational level, but can be daunting for scientists who have become accustomed to a different way of doing things. “I believe it is important to do the right thing by the scientific community. The proposed standards are designed to ensure that published qPCR results are meaningful, accurate, and provide researchers with sufficient information to faithfully reproduce results,” says Shirley Welsh, senior director PCR Systems, Life Technologies (Carlsbad, Calif.).
One way that Life Technologies is enabling customers to meet MIQE guidelines is through their real-time PCR software. Says Welsh, “The researcher will have the option to export data in a RDML format and utilize such nomenclature called out by the MIQE guidelines as Cq.” The Cq, or quantification cycle, replaces the older term cycle threshold (Ct). RDML, or real-time PCR data markup language is a universal data standard for sharing qPCR results. The development of RDML has been a separate effort from the development of MIQE guidelines, but both function in synergy with each other, and have been created by some of the same scientists. RDML developers Jan Jellemans and Jo Vandesompele, co-founders of Biogazelle, a real-time PCR data-analysis company based in Ghent, Belguim, are authors on the 2009 Bustin, et al. MIQE paper.
The RDML data standard is a flat XML file that allows the annotated qPCR data to be exchanged between bundled instrument software and third-party data analysis software, as well as between scientists at different workstations or in different locations. With the advent of online supplementary materials in peer-reviewed journals, this data can also be made available between journal authors, reviewers, and readers. Bio-Rad also is supporting MIQE compliant data analysis in their qPCR software packages. Recently, Bio-Rad entered an arrangement with BioGazelle to distribute qbasePLUS with our CFX systems, a program that enables users to annotate their experiments with MIQE compliant experimental details and supports export of data in the RDML format.
MIQE guidelines have not been embraced as widely or as rapidly as its originators had hoped. MIQE author, Stephen A. Bustin, PhD, Professor of Molecular Science, Queen Mary University of London, has restated the case for MIQE in a new publication on qPCR publications.2 For a period from May 2009 through August 2009, 700 scientific publications mentioned qPCR, but only 23 of these cited Bustin, et al. “After nine months, the impact has been disappointing. The major journals are continuing to produce papers that use qPCR inappropriately. Something really needs to be done about this.”
About the Author
Catherine Shaffer is a freelance science writer specializing in biotechnology and related disciplines with a background in laboratory research in the pharmaceutical industry.
1. Bustin SA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611-22.
2. Bustin SA. Why the need for qPCR publication guidelines?-The case for MIQE. Methods. 2009; Dec 16. [Epub ahead of print]
This article was published in Drug Discovery & Development magazine: Vol. 13, No. 3, April 2010, pp. 32-34.