Background

Real-time quantitative PCR (qPCR) is a cornerstone technique in molecular biology that enables highly sensitive profiling of gene expression levels [1]. Application areas of qPCR span diverse fields of research, ranging from basic biological investigations to clinical diagnostics. In addition to assessing mRNA expression profiles, qPCR is a robust method for detecting DNA copy number variations (CNVs). While both absolute and relative quantification strategies can be used, relative quantification is more commonly used in qPCR analysis due to the inherent complexities and specific requirements of absolute quantification, which often limit its utility in specialized applications such as virus quantification. The ΔΔCq (ΔΔCt) method commonly used for relative quantification in qPCR analysis robustly compares mRNA transcript levels and CNVs [2]. This method determines relative gene expression by first normalizing the target gene’s Cq value with respect to those of reference genes, then rescaling the normalized value to a control group. This procedure is crucial for accurately interpreting biological changes in gene expression and for CNV analysis.

Despite its widespread adoption and critical importance for relative gene expression and CNV analysis, the ΔΔCq method often presents significant hurdles for wet-lab researchers in terms of accessing and utilizing appropriate analysis software. Many available software solutions are proprietary, commercially licensed, platform-specific, or require a certain level of computational skills and programming knowledge, which can be a barrier for many experimental biologists. For these researchers, alternative solutions such as spreadsheet-based qPCR analysis are often error-prone and time-consuming for multiple genes, whereas advanced statistical software can be unnecessarily complex for routine tasks.

To overcome these limitations, we developed a user-friendly tool for qPCR data analysis: Click-qPCR. Unlike many existing solutions that may require local software installation, command-line operations, or a foundational understanding of programming environments, Click-qPCR is a web-based application that requires no setup beyond a standard internet browser. This tool is designed to provide a user-friendly, intuitive, and readily accessible web-based platform specifically designed for rapid and interactive analysis of qPCR data. The application can instantly process typical qPCR datasets, such as those from a 384-well plate, making it ideal for routine high-throughput analyses. Its focus on operational simplicity and immediate visual feedback makes Click-qPCR ideal for researchers, students, and technicians with limited bioinformatics experience.