New, Automated Path to Ask More Complicated Questions and Receive Enhanced Answers
Dr. Sachin Katyal, University of Manitoba and CancerCare Manitoba
With more than a decade of experience, few researchers have more expertise than Dr. Sachin Katyal when it comes to the comet assay, also known as single cell gel electrophoresis (SCGE). As a post-doc at St. Jude Children’s Research Hospital, Dr. Katyal incorporated this assay to study neural damage, and now in his dual role as Assistant Professor in Pharmacology and Therapeutics at the University of Manitoba and Senior Scientist with the Research Institute in Oncology and Hematology (RIOH) at CancerCare Manitoba in Winnipeg, Canada, Dr. Katyal applies the comet assay in his studies on brain tumors and other cancers.
The comet assay principle involves embedding exposed cellular DNA in an agarose gel. When an electric current is applied to the gel, any damaged or fragmented DNA will migrate at a faster rate through the gel compared to intact DNA. Once fluorescently stained and imaged, the result visually resembles the assay’s celestial namesake, with a comet “head” containing intact DNA, and a comet “tail” of fragmented DNA. “It’s a very simple yet powerful technique, but it requires significant time and patience,” notes Dr. Katyal. “Making the comet slides is intensive enough, but the subsequent manual analysis, clicking and counting individual comets under a microscope, is ten times more exhaustive, and user bias can become an issue.” In fact, one day of comet assay lab work could lead to weeks of manual analysis.
High-throughput comet assay manifolds already exist to increase the number of samples processed per run, but Dr. Katyal wondered if it would ever be possible to truly automate the comet assay analysis along with the processing. With that thought lingering in the back of his mind, he attended a BioTek Instruments seminar hosted by his colleague, Dr. Kirk McManus, Associate Professor in the Department of Biochemistry and Medical Genetics at the University of Manitoba. During the seminar, the Cytation™ 5 Cell Imaging Multi-Mode Reader was discussed and demonstrated, prompting Dr. Katyal to ask if the comet assay could be automated. BioTek’s Applications Laboratory experts were brought into the discussion, and based on Dr. Katyal’s criteria, including that for calculating the comet tail moment, which he considers the most credible and accurate measurement of genotoxic damage, new analysis algorithms were created in Cytation 5’s integrated Gen5™ Microplate Reader and Imager Software, along with a specially-created slide adapter to accommodate CometAssay® slides from Trevigen® (Gaithersburg, MD).
Now, instead of individually imaging and manually analyzing comets, Cytation 5 can image every well in a 96-well comet assay plate, and process multiple plates each day as each 96-well slide can be imaged in approximately 30 minutes. Then, the software can automatically analyze every single comet to determine the % DNA in the comet tail and comet tail moment, two parameters commonly used to quantify DNA damage. Analysis time is reduced to merely an hour, and user bias and fatigue are completely removed. Dr. Katyal’s lab also incorporates an EL406™ Washer Dispenser to facilitate and automate slide preparation, further reducing user error and processing time.
BioTek and Dr. Katyal collaborated further on another genotoxicity assay, called gamma H2AX. This and the comet assay are complementary genotoxicity assays used to study mechanisms of cancer chemotherapy as well as classical DNA repair biology. In the γ-H2AX indirect measure of genotoxicity, damaged DNA triggers a response mechanism that rapidly phosphorylates H2AX to γ-H2AX. A feedback loop creates multiple phosphorylation events localized on a histone adjacent to the DNA break, creating nucleation sites, or punctate foci in the cell’s nucleus. These foci are easily visualized when bound to an antibody, yet the manual counting and analysis time is also laborious and prone to user error. In automating γ-H2AX analysis, Cytation 5’s Gen5™ software incorporates a dual-mask method. In each well image, the software automatically places a mask around each nucleus. Then it determines areas of increased pixelation, and refines to those that are foci, for a final calculation of number of foci per nucleus. Cytation 5 is also used for biochemical assays, and shared among other labs in the department.
“Cytation 5 revolutionized how we do a lot of our assays,” says Dr. Katyal. “Every time I show this to a fellow DNA repair biology colleague, they fall off their seats, saying, ‘That’s fantastic!’” He is spotlighting the automated, high-throughput assays in recent talks, and notes that is helping him to increase his professional network on an international stage, and form new collaborations. The partnership between BioTek and Dr. Katyal continues to grow and expand as new capabilities and new assay platforms are explored. He comments that this collaboration will ultimately help him and other researchers to, “ask more complicated questions and get better answers”, both from a research standpoint in DNA repair biology, and as it applies to a clinical setting to detect cancer resistance and enhance drug efficacy.
“It’s not just the technology that makes this academic/industry partnership work,” he notes. “It’s the know-how and the people behind it.” He cites his expertise as amongst the first researchers to apply the comet assay to neuronal DNA damage studies, and BioTek’s expertise in microplate technology, along with their service and support. He appreciated BioTek’s willingness to dedicate resources to visit the lab for initial setup and training, and also to continue to assist with assay optimization and other collaboration opportunities. He also notes that the students and other members of his lab are now able to develop an expertise that they can apply to the lab’s research goals and also take with them as they build their own careers.
For application detail on the collaboration between Dr. Katyal and BioTek, visit:
Thanks to Dr. Sachin Katyal for sharing his BioTek experience.