MIQE guidelines for future publications on qPCR

By Dr. Marcus Neusser, European Product Manager Gene Expression, Bio-Rad Laboratories

In 2009 Stephen Bustin, together with some renowned scientists - all experts on quantitative real-time PCR (qPCR) - published a paper in Clinical Chemistry recommending a set of guidelines with some essential (59) and some desirable (28) check points for documentation of the minimum information necessary for evaluation of qPCR experiments (MIQE) 1. The paper emphasises guidelines to encourage better experimental practice, allowing more reliable and unequivocal interpretation of quantitative PCR results.

Background

In the past there was only a limited awareness that, for example, sample extraction as part of the gene expression workflow using real-time PCR technology, is just as important a determinant of the final result as the PCR itself.

Researchers working on gene expression analysis are often dealing with two major problems:

1. The biological issues related to careful sample selection and data interpretation
2. The technical issues of sample extraction, replicates, normalisation etc.

Jo Vandesompele has provided a solution to the normalisation problem, while Michael Pfaffl addressed PCR efficiency questions and Anders Stahlberg and Mikael Kubista highlighted the challenges related to the RT-step.2, 3, 4, 5  However, in the literature, still many contradictory and conflicting results can be found. In one serious case, a publication in Lancet in 2002, wrong interpretation of RT-qPCR results indicated an association between measles virus and bowel pathology in children with developmental disorders. The Lancet paper had to be retracted as the conclusions supporting that link had nothing to do with the results that the authors had actually obtained. Ongoing debates among scientists on these sorts of issues and increased awareness resulted in the publication of a set of best practice guidelines to conduct and report qPCR experimental results (MIQE).

The MIQE community consists of academic and commercial researchers and indeed anyone who is interested can contribute to its discussions. Companies, like Bio-Rad, have also contributed to the cause of fostering awareness: Bio-Rad's European qPCR Conference series in 2009 was dedicated to MIQE with speakers such as Jan Hellemans, Kim De Leeneer, Michael Pfafl, Jo Vandesompele and Steven Bustin presenting.

In the future more commitment is expected from many journal editors, as the quality of papers today in terms of the information provided is no better than it was 10 years ago according to Stephen Bustin. Even in high impact journals, information is often inappropriate and may even be misleading.

Some researchers have articulated that the MIQE guidelines are too restrictive and far away from laboratory reality. However, qPCR and RT-qPCR are not simple assays and the MIQE guidelines reflect the complexity of the assays themselves - a major aim of the MIQE guidelines is to help researchers to design better assays.

Controversies and concerns

Primers and Probes:

One important issue concerns the requirement to publish primer and probe sequences, as small changes in primer and probe sequences, enzymes and experimental conditions can result in varied results. Some vendors are providing minimal information with respect to their assays, but the MIQE community go further by suggesting that publications of all primer sequences are essential and probe sequences are desirable. It recommends that researchers who wish to use pre-designed assays should buy from companies that comply with the MIQE guideline. Researchers may also use other sources of information like the PrimerDB database, to get validated assays. This database is freely available with access to other researchers' experience and assessments 6.

Normalisation:

The normalisation procedure for mRNA and Micro-RNA has been addressed repeatedly by different methods, for example by Jo Vandesompele's Genorm standard. Unfortunately most researchers still do not normalise. This may be due to the fact that no consensus has been established on how best to normalise. It is still an area of active discussion and characterised by numerous publications.

PCR efficiency:

Theoretically the PCR efficiency should be 100% and the assay design should always aim to be as close to it as possible using a dilution curve for validation. However, there are some other interesting approaches and debates, contributing numerous solutions to this topic. Some software is available that provides both a conventional threshold analysis mode and a "regression" method, so that researchers can decide on their preferred analysis method.

Integrity of RNA:

Lots of researchers still don't look at RNA integrity or inhibition. There seems to be a controversy as to whether these critical data should be reported. However, RNA degrades easily and requires continuous monitoring for its quality. Additionally, as reported by Anders Stahlberg and Michael Kubista, if you use different reverse transcriptases you get different results and also the cDNA priming strategies have an impact on the results. Researchers should consider these facts in their experiments.

The Real-time PCR Data Markup Language (RDML):

The MIQE community is promoting the use of common terminology in qPCR, such as Cq (cycle of quantification) so that there is a consistency in understanding amongst researchers. The Biogazelle qbasePLUS Software is, to date, the only fully MIQE compliant software in terms of language and its reporting of data. Bio-Rad offers its CFX real-time PCR system with a free licence of the Biogazelle qbasePLUS software supporting the idea of RDML mark-up language.

Conclusion

When asked recently in an interview how MIQE changed his daily life in the lab, Steven Bustin explained to Bio-Rad: "In 1997 I remember coming into the lab every morning and being fascinated with the amplification plots. Over the years we simply learned to develop protocols that weren't in every respect MIQE compliant but, together with colleagues' expertise and experience, evolved into MIQE. Applying the MIQE guidelines may impact the laboratory's workflow as it reflects a best practice at the time. In the past researchers did lots of things wrong because they did not know any better. Today following the MIQE guidelines is surely better than having to retract papers, like the MMR case. Applying MIQE guidelines will save both time and money, as it will result in the elimination of wrong and misleading publications."

(To request the full printed version of the interview with Stephen Bustin, e-mail uk.lsg.marketing@bio-rad.com)

For more details on MIQE please visit: http://www.rdml.org/miqe.php

Literature:

1 Bustin et al., 2009  Clinical Chemistry, Vol. 55:4, 611-622

2 Pfaffl, 2001, Nucleic Acid Research Vol.29

3 Stahlberg et al., 2004, Clin. Chem. Vol.50, 509-515

4 Kubista et al., 2006 Molecular Aspects of Medicine Vol. 27, 95-125

5 Vandesompele et al, 2002, Genome Biology Vol. 3

6  Pattyn et al., 2003 Nucleic Acids Research, Vol. 31, No. 1