Spike-in normalization is a technique that aids molecular biologists in ensuring the precision of their findings, though it is prone to misuse. A recent study has proposed new guidelines to enhance the effectiveness of this important method.
A team of researchers at the University of California San Diego has introduced new recommendations aimed at helping scientists improve their results when measuring the interactions between DNA and proteins. Gaining insight into these interactions is vital not only for comprehending human biology as a whole but also for aiding in the development of new treatments for various diseases, including several types of cancer.
The research team concentrated on spike-in normalization, a commonly used technique in molecular biology that ensures results are both accurate and dependable. This method involves introducing a known amount of chromatin (the complex of DNA and associated proteins) to a sample before analysis. This addition allows researchers to adjust for variations across multiple samples being compared. Spike-in normalization is especially beneficial when evaluating two different conditions, such as assessing the effect of a drug by comparing treated versus untreated samples, or analyzing mutations or deletions in essential genes by comparing normal versus mutant samples.
By analyzing publicly available datasets that employed spike-in normalization, the researchers pinpointed frequent instances of misuse and identified opportunities to enhance the technique by incorporating additional quality control measures, often referred to as “guardrails.” Through reanalyzing this data and performing their own experiments, the team formulated a list of nine crucial recommendations for researchers utilizing spike-in normalization, which could significantly enhance the integrity of their findings. These recommendations cover ensuring consistent quality control practices, adhering to established best practices in computational analysis, and validating results with supplementary analytical methods.
“Numerous studies apply spike-in normalization, and our findings cast doubt on the biological interpretations derived from this approach,” stated senior author Alon Goren, Ph.D. “Our suggestions can help address some of the challenges associated with spike-in normalization, allowing us to continue benefiting from this important technique.”
The study, published on September 13, 2024, in Nature Biotechnology, involved contributions from Lauren Patel and Yuwei Cao at UC San Diego, alongside Eric Mendenhall, Ph.D. at the HudsonAlpha Institute for Biotechnology. The research was co-led by Alon Goren, Ph.D., and Christopher Benner, Ph.D., both serving as associate professors in the Department of Medicine at UC San Diego School of Medicine.
