Do Your RT-qPCRs Make The Grade?
Real-time PCR is a technique that is now commonly employed in almost all molecular biology laboratories to quickly answer very specific questions. Northern and Southern blotting are now a thing of the past. No longer do we wait days to know whether a gene is expressed. We can have the answer in 45 minutes!
But with the widespread use of such a wonderful and sensitive technology comes differences in how results are reported in the literature. There are also differences between reviewers reading these papers and their understanding of the essential information required to judge the accuracy of the reported data.
To overcome this increasing problem of lack of consistency in publications, a panel of real-time PCR experts published a set of guidelines containing what they consider the minimal information required when reporting qPCR results. That paper called The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, was published February 2009 in the Journal of Clinical Chemistry.
This is not only a great resource for authors, but it also essentially a troubleshooting guide as well. If you don’t have an answer to each of the item on the checklist, then maybe you are missing an essential piece of information in your experiment.
Standardization of nomenclature:
To help standardize the lingo used in real-time PCR, the authors start off by clarifying common terminology.
- For example, the term RT-PCR is reserved for reverse transcription-PCR while the term RT-qPCR should be used for quantitative experiments. The RT in RT-PCR does not stand for “real-time” in this case. The q designates that the experiment is a real-time PCR experiment. So it is qPCR or RT-qPCR when the experiment invoves real-time methodology for DNA or RNA, respectively.
- In regards to the use of genes for normalizing qPCR experiments, the authors recommend the use of the term reference genes and not housekeeping genes.
- TaqMan probes should be called hydrolysis probes which is in contrast to hybridization probes of which FRET and Molecular Beacons are a class. TaqMan is a trade name for hydrolysis probes and is not a scientific term.
- Several terms are used to describe the point at which fluorescence becomes detectable and measurable. These include the threshold cycles (Ct), crossing point (Cp), and take-off point (TOP). The authors explain that these are marketing terms. They propose the use of the word quantification cycle (Cq) as the universal word for the cycle where fluorescence is measured.
The Checklist:
The checklist for the essential and desirable information for a publication is quite long and may be intimidating to researchers who may have not realized how much information is really critical to judging the scientific validity of their work.
But in truth, adding this information to your paper will only help make your conclusions stronger and make it more difficult for reviewers to reject your results. Topics that need to be addressed include the experimental design, the method of RNA or DNA extraction and quantification, qPCR target information, kits used, efficiency and slope information, and data analysis information including NTC information, normalization method, and repeatability.
It is truly a great checklist for making sure your paper gives a complete story.
Who should read this paper:
If you are using qPCR or RT-qPCR in your project, then read this paper. And if you are reviewing papers using qPCR, you should read this paper.
Too many critical details are being left out of papers and conclusions are being made that may not necessarily be correct. What if someone else’s paper scoops your work because it was not reviewed as critically as yours? And then the data is not even correct?
What if you are trying to repeat someone else’s work and you waste valuable hours and expensive reagents and can’t replicate the results because information was missing?
Ideally, all papers need to be reviewed for the same criteria and with the same scrutiny before being accepted for publication. This type of standardization provided by the MIQE guidelines are meant to ensure fairness during the review process and make sure that only high quality results make it to print.
What do you think about the MIQE guidelines? Do you like them? Will you follow them? Tell us your thoughts.
Digg
Stumble
Reddit
Delicious
TwitterAbout the Author
Suzanne Kennedy
Suzanne is Director of R&D at Mo Bio Laboratories in California, and the author of their blog, The Culture Dish. She has a PhD in Microbiology and Immunology from Virginia Commonwealth University.
I hate acronyms. Too many times you come across a new PCR acronym that you only need remember for the length of the paper. Then, never to see it again. Still, probably alone here. I don’t like the use of ‘q’ since this denotes quantitative PCR which is still being done without real-time methods. Then of course you have diagnostic yes/no real-time PCR assays (which we do) where we are not quantifying !
One picky thing Suzanne. FRET is the principle of the probe chemistry (largely all the probe chemistries I think?) – not a particular one. You are referring to the LightCycler HybProbes?
I think the guidelines are good – although I admit I got the paper when it came out and haven’t read it properly yet!
Hi John,
Yes. FRET is a generic mechanism. The authors recommend that when talking about LightCycler probes, that the term “dual hybridization probes” is used (point 1.4).
Best,
Suzanne
John,
I would point out that when you use a real-time qPCR instrument in plate read mode, the assay is no longer a real-time assay in the way the nomenclature was intended. Surely, you are collecting the data right then an there but the time component is very short in plate read mode. Thus, I would argue that the real-time moniker does not apply to plate read assays. In that mode, the instrument could be substituted with any fluorescence plate reader, if there were software to analyze the resulting data.
I see we are on the same page when it comes to the term quantitative to denote these assays. You are quite correct that there was quantitative RT-PCR and PCR prior to the introduction of the real-time instruments. Further, there are other methods currently in use to quantify transcripts and DNA targets that do not utilize a real-time instrument, the b-DNA technique for example. Thus, whenever a paper is referring to a real-time qPCR experiment, I have always demanded that they spell that full term out (real-time qPCR) in the first instance of the paper before shortening it to qPCR alone. Folks tend to be lazy in their writing and research, which is the reason the hammer has finally come down with the MIQE guidelines.
Lastly, FRET pertains to any two fluorescent dyes that have sufficient spectral overlap such that the second dye can absorb the emitted light from the first when they are sufficiently close to one another in proximity. There are many examples of FRET used with proteins to detect close interactions in the literature. To my knowledge, all the probe chemistries used in real-time qPCR utilize FRET for signal generation.
I hope this is helpful,
Greg S.
Hi Suzanne,
Nice post. This (quality and reproducible qPCR analysis) is something that has also been of some concern over at The Plant Cell and I blogged about it a few months ago.
You can check it here http://amontenegro.blogspot.com/2009/05/plant-cells-struggle-with-qpcr.html
The article in question is Rieu I, Powers SJ. (2009) Real-Time Quantitative RT-PCR: Design, Calculations, and Statistics. The Plant Cell 21:1031-1033 (2009)
Cheers!
I guess one never covers every possibility – I’d be interested in how many are using the plate option for the yes/no calling (only on ABIs I think?). I’d say most diagnostics would use the full real-time function to assess curves and Cq’s etc.
Yes, there are lots of alternatives to PCR. . but I think most of them are going the route of ‘real-time’-style automation. I think bDNA as as example has the ‘plate-read’ version at the end.
There was a very good ‘electronic roundtable’ in the book ‘Real-Time PCR in Microbiology’ ed. Ian Mackay (profess my bias here as a co-author). . but no opinions varied more widely than on the possible real-time PCR acronyms
[...] used as a further claim of increased sensitivity of something like a hydrolysis probe (to be MIQUEy about it) over a dye-based assay such as SYBR Green. Yes, a particular assay may show this [...]
[...] the data is qPCR, then take the time to look even more carefully. According to the MIQE guidelines, the authors need to explain the nculeic acid purification method, yields, and purities, which kits [...]