The MCTOT app: A publicly available tool for statistical cycle-to-threshold analysis and inference of informative but uncertainly determined qPCR data.

As a common experimental technique, qPCR (Quantitative Real-time Polymerase Chain Reaction) is widely used to measure levels of nucleic acids, e.g., microRNAs and messenger RNA. While providing accurate and complete data, researchers have inevitably encountered uncertainly determined qPCR data because of intrinsically low amounts of biological material. The presence of incomplete or uncertain qPCR data challenges interpretation accuracy. This study presents a web application that integrates two sophisticated statistical methods - a flexible regression approach and a two-group hypothesis testing technique - to enhance the accuracy and robustness of qPCR data analysis with informative but uncertainly determined observations. To demonstrate the versatility and efficacy of our MCTOT (Multi-Functional Cycle-To-Threshold Statistical Analysis Tool) application, this study presents two distinct examples employing two-group hypothesis testing. The first example delves into an analysis of pathogens in wastewater, an area gaining increasing relevance for public health surveillance. The second example illustrates an application in the realm of liquid biopsy, a rapidly evolving field in disease diagnostics, monitoring, and early treatment. Moreover, the application's process is further exhibited through another liquid biopsy example, wherein the flexible regression method is employed to detect the hemolysis effect on a molecular target. These examples demonstrate the tool's capacity to not only identify significant differences between groups but also to quantify the effect size, a crucial aspect in biomedical research. The MCTOT web application stands as a pioneering step toward empowering researchers to harness the full potential of qPCR data, especially when dealing with informative but uncertainly determined observations. It also paves the way for further development of web-based tools that adhere to the refined CTOT (Cycle-To-Threshold) methodology, opening new avenues in qPCR data analysis and interpretation. The developed application can be accessed online through Shinyapps.io at https://ctot.shinyapps.io/bioinformatics/ for open access.