Reverse-transcription quantitative PCR (RT-qPCR) has been the subject of considerable controversy. While the technique is considered the gold standard for quantifying gene expression in a cell, there are so many variables involved that two researchers could perform the same experiment and end up with wildly different results. And although a study may produce a statistically significant result, it's hard to know if that result is truly valid or if the data might have been skewed due to a technical error.
Of course, guidelines only work if they are widely adopted by labs and journals and remain up-to-date with new technical developments. So where do the MIQE guidelines stand now, three years after their initial publication?
It’s easy to perform PCR experiments, says Jim Huggett, who leads the nucleic acid metrology research group at UK-based biotechnology company LGC Limited. "But the trouble is, there's an awful lot of upstream preparation needed and a whole multitude of things you can do beforehand which will alter the results you get." If researchers don't consider these factors—including extraction method, sample storage conditions, and more—they may not realize what is influencing the results.
Bustin knows all about results that are not what they appear to be. He investigated studies that used qPCR to show the measles virus was present in intestinal tissue of children with autism, implicating the measles, mumps, and rubella (MMR) vaccine in the development of the disorder. Because the researchers had skipped an important step in the protocols—they had inadvertently neglected to add reverse transcriptase to several samples to convert the measles virus RNA to DNA—Bustin was able to determine that the samples were contaminated with DNA plasmids from a facility located next door to the lab. The positive qPCR signal that the researchers had picked up was from those plasmids, not the measles virus.
Although this well-publicized and extreme case demonstrates the potential usefulness of the MIQE guidelines, many researchers still don't follow them. "We're beginning to see people becoming more careful," says Bustin, "but if you look at the numbers of papers that cite the MIQE guidelines, it's about 7 percent in terms of qPCR, which means that 93 percent of papers are still being published with inadequate information that doesn't allow anyone to decide whether the data are real. And if you look at the data, you can very often see that they are meaningless." For example, when a paper reports a one-and-a-half fold difference in gene expression that is normalized against the expression of a single unvalidated reference gene, it's hard to conclude that the result is valid.
Despite the low percentage of papers citing the MIQE guidelines, one group is taking the guidelines very seriously and advocating that researchers stick to them: the companies that sell reagents and other qPCR supplies. "There's clearly an improvement in the quality of the publications that are being published [as a result]," says Huggett.
In a paper published recently in PLoS One, biomedical researcher Francis Jacob from the University Hospital Basel in Switzerland and colleagues reported their attempts to identify a set of suitable, reliable reference genes for several different human cancer cell lines and to determine whether MIQE guidelines are actually being followed by labs (2). His group found that many of the studies look complete on the surface, but when you delve into the details, you find that important data are missing. Many papers don't report the efficiency of their reference genes or their qPCR data, and only 30–40 percent of published studies that investigated reference genes actually followed the MIQE guidelines.
New Digital Guidelines
Meanwhile, qPCR is ever evolving. Its newest incarnation, digital PCR or dPCR, is being used by more and more labs. As a result, a new set of MIQE guidelines geared to the specific concerns of this brand-new version of PCR have recently been published in Clinical Chemistry (3).
Most researchers in the field concede that the original MIQE guidelines came a bit too late in the process, after years of questionable qPCR data reporting. With the advent of dPCR, Huggett says that the goal was to publish guidelines early on in the adoption of this new version of the technique.
Although it remains expensive and time-consuming, dPCR has gained some traction with the development of microfluidics and nanofluidics as well as with emulsion chemistries used in next-generation DNA sequencing. In addition, dPCR is generally more precise than RT-qPCR, says Huggett. "It's much more reproducible without the need for standard curves," he says. "It has the potential to revolutionize quantification by using molecular methods."
But is it too early in the development of this new technique to publish guidelines? The authors don’t believe so. They reasoned that dPCR does seem to have legs as a technique and that certain dogmatic ideas about the process are already becoming set in stone. "We are very much trying to, at an early stage, get people thinking about the benefits of dPCR, while still remembering there are some limitations, and most importantly, that you need to prove to yourself that the technique is working well enough, rather than just assuming it's fine," says Huggett.
Ultimately, the goal for the qPCR and dPCR MIQE guidelines is for the data to become more transparent, says Bustin. When the data are more standardized, other researchers can analyze them and come to their own conclusions about the validity of the results. "My conversion to this was when I was acting as an expert witness to the MMR trials," says Bustin. "I had data. I had to report on it, and it wasn't clear how they had gotten that data. Once I got access to the raw data and analyzed it myself, I was able to understand how they had gone wrong."
The key, he emphasizes, is access to the raw data, and he believes that, within five years' time, publishing raw data will be the standard procedure for qPCR papers. "If you could import the [raw data] file into your analysis program, then you could see right away whether you believe the data or not. You can immediately come to conclusions about the quality of the work. I think that's where we're headed."
1. Bustin, A., V. Benes, J. Garson, J. Hellemans, J. Huggett, M. Kubista, R. Mueller, T. Nolan, et al. 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55:611-622
2. Jacob, F., R. Guertler, S. Naim, S. Nixdorf, A. Fedier, N. F. Hacker, and V. Heinzelmann-Schwarz. 2013. Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines. PLoS ONE 8(3):e59180+.
3. Huggett, J. F., C. A. Foy, V. Benes, K. Emslie, J. A. Garson, R. Haynes, J. Hellemans, M. Kubista, R. D. Mueller, T. Nolan, M. W. Pfaffl, G. L. Shipley, J. Vandesompele, C. T. Wittwer, and S. A. Bustin. 2013. Guidelines for minimum information for publication of quantitative digital PCR experiments. Clinical Chemistry (April).