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GAINESVILLE, Fla. � A simple yet elegant equation published by a University of Florida professor nearly 25 years ago has earned a place among the most influential scientific works of the century.

The 2001 publication co-authored by , Ph.D., chair of pharmaceutics in the UF College of Pharmacy, has garnered nearly 150,000 citations in the Web of Science database, securing its place as the second most-cited research article of the 21st century and the fifth most-cited publication of all time, .

The paper introduced a now widely used approach for gene expression analysis and was co-authored by Schmittgen while he was an assistant professor at the Washington State University College of Pharmacy. He collaborated with Kenneth Livak, Ph.D., a pioneer in the field of quantitative polymerase chain reaction, or PCR, .

Quantitative PCR is a widely used technique in life sciences, helping researchers measure how genes are expressed in different samples. Before Schmittgen and Livak’s work, scientists often relied on standard curves, an extra step that made the process more complex. The 2−ΔΔCT (spelled out as �2 minus delta delta C-T�) equation, derived by Livak, simplified the calculations, allowing researchers to quickly compare gene expression levels between samples.

“If you wanted to compare the amount of RNA in a tumor versus normal tissue, you would use quantitative PCR to measure those differences,� Schmittgen said. “The equation lets you express those results as a fold-change � for example, showing that a tumor has 10 times more mRNA than normal tissue.�

Schmittgen attributes two key factors to the paper’s immense influence. First, quantitative PCR is used across many life sciences, from cancer research to infectious disease studies, making the method relevant to a broad scientific audience. Second, methodology papers tend to be the most cited in history, as researchers frequently reference them when applying established techniques.

“Methodology papers endure because they remain useful over time. Scientists rely on established methods, and the 2−ΔΔCT equation is one of them,� Schmittgen said. “What this paper did was really demystify the calculations so even a high school student doing quantitative PCR would understand it.�

Schmittgen and Livak’s paper had an immediate impact, earning 50 to 60 citations in its first year � far outpacing any of Schmittgen’s prior research. Since then, thousands of scientists worldwide have referenced their work, with many reaching out to Schmittgen for guidance on applying the 2−ΔΔCT method in their studies. It’s gratifying to help, Schmittgen said.

“I could have never predicted almost 25 years later it would have been cited so many times,� he said. “But it’s reassuring to know that Dr. Livak and I have made a lasting contribution to science.�

A 2003 article authored by , Ph.D., the Hyatt and Cici Brown Chair in Business and clinical professor in the UF Warrington College of Business, ranks No. 17 on Nature's list of the most-cited papers of the century. Published in the , the paper explores methodological biases that pose challenges to behavioral research across disciplines such as psychology, management and related fields.