Current Protocols

Analysis of Reactive Oxygen Species–Induced Cellular Damage in Cervical Cancer

Abstract Reactive oxygen species (ROS) are highly reactive oxygen‐based molecules comprising hydrogen peroxide, hydroxyl radicals, superoxide anion, and singlet oxygen. These species are produced intracellularly and play an important role in cellular signaling and metabolism. Their high reactivity damages intracellular macromolecules such as lipids and DNA. In cancer biology, ROS display a dual role: they promote cancer cell proliferation at low to moderate levels, whereas excessive accumulation overwhelms antioxidant defenses, causing oxidative stress and apoptosis. This has resulted in therapeutic strategies that selectively increase ROS in cancer cells to induce apoptosis. Vitamin D has demonstrated anti‐cancer properties, with one proposed mechanism involving ROS‐mediated apoptosis. This article outlines a workflow to investigate ROS‐induced cellular damage by vitamin D 3 in HeLa cervical cancer cells. The study begins with quantification of ROS levels and assessment of mitochondrial membrane potential in HeLa cultures. Transmission electron microscopy is used to examine mitochondrial ultrastructure. Lipid peroxidation quantifies downstream ROS‐mediated membrane and cellular injury. Antioxidant enzyme activities, including superoxide dismutase and catalase, measure cellular anti‐oxidative defence capacity. Lastly, the role of ROS inhibition of AKT signaling, leading to reduced cell survival and apoptosis, is quantified by immunoblotting. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Preparation of HeLa cell cultures and treatment with vitamin D 3 Basic Protocol 2 : Quantification of ROS‐positive cells using the Muse Oxidative Stress assay Basic Protocol 3 : Measurement of mitochondrial membrane potential using the Muse MitoPotential assay Basic Protocol 4 : Ultrastructural evaluation of mitochondrial damage by transmission electron microscopy Basic Protocol 5 : Evaluation of lipid damage using a human 8‐iso prostaglandin F2α ELISA Basic Protocol 6 : Evaluation of total superoxide dismutase activity using an activity assay kit Basic Protocol 7 : Evaluation of catalase activity using an activity assay kit Basic Protocol 8 : Immunoblot analysis of AKT to assess PI3K/AKT signaling

A Novel NGS‐Based Algorithm for Precise HPV Genotyping and Co‐infection Detection in QCMD Samples and FFPE Tissues

Abstract Accurate genotyping of Human papillomavirus (HPV) is essential for cervical cancer screening, prognostic risk assessment, and epidemiological surveillance. Conventional methods, such as Sanger sequencing and hybridization‐based assays, often fail to detect mixed infections, particularly in cases involving DNA from formalin‐fixed paraffin‐embedded (FFPE) tissues. In this protocol, we present an optimized next‐generation sequencing (NGS) based pipeline for comprehensive HPV genotyping. Our workflow includes a curated HPV reference database of 229 HPV genotypes and a proportional read‐mapping algorithm. We validated our method using 30 samples from the Quality Control for Molecular Diagnostics (QCMD) proficiency testing panel and were able to fully replicate the expected outcomes—including detecting the defined co‐infection with HPV16/18. We then applied our approach to 50 clinical FFPE samples, identifying 44 HPV‐positive samples, including a co‐infection scenario that traditional diagnostic assays might have missed. This low‐cost, open‐source NGS‐based pipeline improves the sensitivity and accuracy of HPV detection from archival FFPE tissue specimens, cervical screening specimens, and other fresh tissue, and successfully closes the gap between research applications and clinical usage. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : DNA isolation Basic Protocol 2 : PCR amplification Basic Protocol 3 : NGS Basic Protocol 4 : Data analysis

High‐sensitivity Epimutations Testing with MS‐HRM

Abstract Epimutation is defined as any heritable change in gene activity that does not affect the actual sequence of DNA. One of the most researched epimutations is promoter methylation of breast cancer susceptibility gene BRCA1 . This epimutation may arise de novo in somatic tissues, such as breast or ovarian cancer tissue, but has also been shown to be widely distributed throughout tissues. BRCA1 methylation detectable in peripheral blood has been associated with the risk of both breast and ovarian cancer in patients without a germline pathogenic variant status of this gene. The strength of this association suggests that diagnostic screening for the BRCA1 epimutation should be considered with potential implications in cancer risk assessment. However, the detection of BRCA1 epimutation may be challenging, as it is generally detectable at a very low level in blood. Methylation‐sensitive high‐resolution melting (MS‐HRM) was shown to provide a high sensitivity of methylation detection, and as it is a PCR‐based method, epimutation screening based on MS‐HRM is labor‐ and cost‐effective. Here we describe the MS‐HRM protocol for BRCA1 epimutation screening. © 2025 Wiley Periodicals LLC. Basic Protocol : High sensitivity constitutional epimutations testing with MS‐HRM