MIQE guidelines : publication or almost a standard?

 I talked about the MIQE guidelines previously, so I am going to give my opinion.
 
MIQE is a publication written by most active scientists about qPCR in the molecular biology planet. Among them, Stephen Bustin, Mike Kubista, Mike Pfaffl, Greg Shipley (great contributors of PCR yahoo forum eg) or Jo Vandesompele.

MIQE is for "Minimum Guidelines for publication of Quantitative real-time PCR Experiments", that is to say a sort of little guide of how is better to publish qPCR results and experiments in order to respect what is a publication or presentation : being able to discuss and reproduce it.

More and more qPCR papers are published and it can be seen from time to time new acronym coming from nowhere or definitions that can be discussed about used techniques being sometimes a bit confusing...

The approach is interesting, but before reading this document, I would like to say that a french standard does exist (followed by other french ones and ISO also) and deal with qPCR : working on Legionella detection but can be read in a second level for all qPCR users because treating results about performances and not only a recipe for Legionella detection only. Named NF T90-471, it has been followed by others like U47-600 working on veterinary samples or ISO 12869, largely inspired from french standard for Legionella.






Back to MIQE.
As said before, the approach has the will to formalize a little bit the exchanges between all qPCR users and also publisher. One of the best reason to me that this text has a real interest was the confusion occuring for a while for the acronym RT-PCR. Indeed, RT would mean "Real-Time"", so quantitative PCR, but also "Reverse Transcription" for PCR using RNA and needing a reverse transcription step to work. This confusion was real for a time among discussion and talks  when people were talking about RT-PCR but thinking about the two different methods. It has lead to real mistake...

This trivial example is not the  main part of MIQE guidelines. This one try to overcome several things like the previous example :

Few things are fixed like :

- "RT-qPCR" for Reverse transcription quantitative PCR and "qPCR" for quantitative PCR.
- "TaqMan probes " are "hydrolysis probe"
- "Housekeeping genes" are"reference genes"
- "FRET probes " are "sondes d'hybridation double"
- "Ct" or "Cp" or "TOP" (not often used) are "Cq" for quantification cycle

Then, a reminder of essential steps to validate a qPCR system is made :
1 - sensitivity and LOD
2 - specificity
3 - accuracy
4 - repeatibility
5 - reproducibility

PCR is not the only point, another important one is done about getting a good quality sample and standards considering microorganism strain to use, extraction and purification protocol.

Another essential step is the standard quantification. Using spectrophotometry (260/280 ratio) or fluorimetry (with intercalant) is recommended to insure final results and verify what is written on the tube tag which can be wrong or slightly diffierent when we talk about biological smaple.


There are many details in this paper, one is very trivial but I think nowadays not so useless : give all the informations to be able to reproduce experiment in another lab. This can be surprising in a science paper, because it should be the basis of any paper in science. But I agree this has to be reminded because I can quite often read papers where few informations are missing or it is written "following supplier protocol". Except that from time to time, supplier protocol is slightly modified but not written in the paper... and when it is tried to reproduce it, it does not work obviously...as a secret.

In short, this paper is very laudable but is no more than a guide for "how to write a qPCR paper". Is it a sign of any drift?
To conclude, this paper is very useful for anyone wanting to publish or to be able to criticize any results using PCR. But standards are also published and even different, this can lead to dual purpose for several part of the paper between MIQE and qPCR standards. As for me, changing Ct by Cq is not very relevant...


Have a good PCR then and see you next time...