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一个单篇文章被引用4,137 次的作者的访谈（生物化学方向）
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[table][tr][td][table][tr][td=1,2] [/td][td]Dr. Kenneth Livak & Dr. Thomas SchmittgenA Featured Paper from Essential Science IndicatorsSM [/td][/tr][tr][td]According to Essential Science Indicators from Thomson Reuters, the paper "Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method," (Livak KJ and Schmittgen TD, Methods 25[4]: 402-8, December 2001) is ranked at #3 among Biology & Biochemistry papers published in the past decade, with 4,137 cites. The paper's authors, Dr. Kenneth Livak and Dr. Thomas Schmittgen, recently spoke with ScienceWatch.com correspondent Gary Taubes about this paper and its impact on academia and industry. [/td][/tr][/table] Dr. Livak is currently in Research and Development at the Fluidigm Corporation in South San Francisco. Dr. Schmittgen is an Associate Professor at the Ohio State University College of Pharmacy. [table=98%,#f0f0e0][tr][td]Below, the paper's authors, Dr. Kenneth Livak and Dr. Thomas Schmittgen, talk with ScienceWatch.com correspondent Gary Taubes about this paper and its impact on academia and industry. [/td][/tr][/table]  Ken, what motivated the work that led to the 2−ΔΔCT method for real-time PCR and your 2001 Methods paper? Livak: In 1994, I went to work for Applied Biosystems, which had just started this project on real-time PCR. I was part of the team there that developed the first instrument for doing it. These projects were multidisciplinary in that they had engineers, chemists, biologists, etc., and I took the lead position on the molecular biology part in terms of developing assays, optimizing assays, and getting detection with the probes to work well. Because Applied Biosystems was introducing a whole new system, they needed documentation to teach their customers how to deal with the results. We provided a document called User Bulletin Number 2. I was the main person involved in drafting that User Bulletin, collecting all the data, and getting it published. [table=98%,#ffffff][tr][td] [table=98%,#c0c0c0][tr][td]
"The number of publications that cite real-time PCR has probably been growing exponentially over the past decade." ~Thomas Schmittgen [/td][/tr][/table][/td][/tr][/table]This bulletin addressed the problem of quantitation by real-time PCR, which is different conceptually than the way people usually think about quantitation. Naively, they think you do a fluorescence measurement and the amount of fluorescence will tell you how much analyte is there. But what you get out of real-time PCR is really more of a kinetic measurement. You do your quantitation based on how many cycles it takes to reach a certain fluorescence threshold. So the whole way you do the quantitation was different than the way people were thinking about it in the past. It really required some basic mathematics background so people could readily take the real-time PCR data and get quantitative results out of it. If this started as a User Bulletin for an Applied Biosystems product, how did you two come to collaborate on the Methods paper? Schmittgen: Well, Ken developed this method and published it in the User Bulletin. So when you purchased the instrument from Applied Biosystems, that User Bulletin would come with all the various manuals. This was pre-internet days, so it was mostly only available to people who had the instrument. Anyway, that was published, but it was not in the general literature. Early on, when people needed to cite something in a paper, they would have to cite the User Bulletin from the company. I was working on a paper for Analytical Biochemistry and I, too, cited the Applied Biosystems User Bulletin, and one of the comments that came back from the reviewers was that it was not appropriate to cite a User Bulletin in the literature. It just so happens that at the time I was editing a thematic issue for the journal Methods on quantitative real-time PCR. I said, "Why don’t I publish Livak’s derivation of the equation in this journal? That way it would be available to everyone in the literature." I had never met Ken, but I called him up, told him what I wanted to do and he agreed with it, and that’s what we did. Livak: You have to realize that when you’re working at a company like Applied Biosystems, there isn’t quite the same incentive to publish in academic journals that there is in the academic world. It’s a good thing to publish, but it’s kind of secondary. It’s not publish or perish in my world. We were interested mainly in designing the instruments and the reagents and developing the methods. Until Tom called, I just wasn’t motivated to submit this to a scientific publication. With Tom offering to help, it got me over the activation barrier of doing the work necessary to make this an academic publication. Naïve question perhaps, but what’s the difference between real-time PCR and not real-time, or regular PCR? Schmittgen: Regular PCR amplifies the RNA or DNA in a sample, and real-time PCR amplifies it and quantifies it. You’re actually measuring something as it occurs in real time. Following the PCR, you get a number, and that number tells you how much RNA or DNA is in the reaction. How hard was it to come up with the mathematics and make this work? Livak: Developing the real-time PCR probe and the system itself was the hard part, and that’s what enabled this technology to become widespread. The mathematics does take careful thought, but our primary advantage was that we were the first ones coming out with the system and the first ones doing the math. A lot of people could have done what we did; we just got there before them. What issues of real-time PCR does this paper address or solve? Schmittgen: What it does, in its simplest form, is allow you to essentially work with the data that is generated from the experiment. You’re getting numbers from the computer that’s attached to the instrument. The question is, what do you do with these numbers? How do you use them? How do you present the data? What Ken’s equations do is allow you to present the data in a way that makes sense of it. Basically what you’re doing is looking at the gene expression, the RNA; you take a cell, treat it with a drug; take another cell, use it as the control, and then you measure the amount of RNA in both cells. This equation then allows you to relate the expression of genes in the treated cell to the untreated cell. What Ken had done very elegantly was to come up with the equations and various assumptions that allowed him to derive the equation. It was a beautiful piece of mathematics. Part of the reason why the method is so popular is that it’s very simple and yet based on this theory of PCR amplification. Livak: The reason that the method—this 2−ΔΔCT method—works is actually pretty interesting. Whenever people do the mathematics for these sorts of things, you have to make all these assumptions so that you can get a relatively straightforward mathematical expression. It turns out that the actual process of PCR is much more complex than these assumptions would indicate, but the simple approximation that we made in the paper still manages to give you very good data. That’s fascinating to me. How is it that these mathematical expressions can sometimes be very useful, even though we’ve come to understand the underlying processes are far more complicated? It turns out that our original assumptions may not be precisely accurate, but there’s still something about the overall process that enables the equation to provide good quantitative results. How much did the Methods article actually differ from the User Bulletin? [table=98%,#ffffff][tr][td] "It turns out that the actual process of PCR is much more complex than these assumptions would indicate, but the simple approximation that we made in the paper still manages to give you very good data." How did the writing process go, considering you guys had never met before and didn’t even know each other until Tom’s phone call? Livak: We basically started with the User Bulletin, and then Tom put that into the form of an academic paper, the way he saw it. Then I worked on writing some of it and having it flow as a paper. Tom added material, including some additional experiments. So he did the first draft based on my User Bulletin, and we then collaborated from there to get it to final form. Are you surprised at how influential your method has been and how remarkably highly cited the Methods paper has become? Schmittgen: I’m very surprised. As I said, my only intention was that I was working on this journal issue and I thought it would make sense to include this paper in the issue and give investigators like myself something in the archival literature to cite. I had no idea it would go so far. Livak: I’ve only recently appreciated the place this real-time PCR work has taken me. When you’re in the trenches working on it, getting the system up and out there, you have one view. And once you introduce these systems, it takes a certain amount of time before they start getting used widely and really start having influence. It’s only over the past two or three years that I’ve become aware how pervasive real-time PCR has become. In that context, I’m not that surprised the paper has been cited a lot. What’s in the paper is pretty basic and fundamental to the technology. It’s really the foundation of how you do things in real-time PCR; it’s the sort of thing that people would refer back to, the place you start if you want to get meaningful quantitative results. How much has real-time PCR changed since 2001? Is it much more sophisticated? Livak: Actually, not in any fundamental sense. What’s changed is that people are doing things faster, and with higher throughput. The actual underlying technology—how you set up the reactions and do the fluorescence measurements—is still fundamentally what it always was. Schmittgen: One thing that’s changed is that it’s become far more popular to use the technology. The number of publications that cite real-time PCR has probably been growing exponentially over the past decade. And more people are using the technology to study more genes. This is the high-throughput aspect of it. Instead of just looking at one gene now, people are looking at hundreds.  Kenneth J. Livak, Ph.D. Fluidigm Corporation South San Francisco, CA, USA Thomas D. Schmittgen, Ph.D. College of Pharmacy Ohio State University Columbus, OH, USA [table][tr][td=2,1]Dr. Kenneth Livak & Dr. Thomas Schmittgen's most-cited paper with 4,137 cites to date: [/td][/tr][tr][td][/td][td]Livak KJ and Schmittgen TD, "Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method,"Methods 25(4): 402-8, December 2001. 4,137 cites. Source: Essential Science Indicators from Thomson Reuters. [/td][/tr][/table] Keywords: real-time PCR, assays, instrumentation, collaboration, RNA, DNA, PCR amplification. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Livak KJ, Schmittgen TD. http://www.umich.edu/~caparray/Files/LeivakddCt_paper.pdf [/td][/tr][/table] [ Last edited by hjlyyc on 2008-7-1 at 19:20 ] |
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