Methods to Quantify the MicroRNA and Study Their Role in Genotoxic Stress

MicroRNAs (miRNAs) are regulatory RNA molecules that adjust the levels of proteins in cells to maintain cellular homeostasis during essential cellular events or stress conditions. A large array of studies are available in the literature, demonstrating the role and regulation of miRNAs in cellular responses to environmental toxicants such as heavy metals, pesticides, and air pollutants. Genotoxicity, the damaging impact of various environmental and chemical agents on DNA, plays a significant role in altering gene expression and affecting cellular health. It is crucial to understand the interplay between the expression of miRNAs and the regulation of gene expression in response to genotoxic insults. Dysregulated miRNAs can lead to oxidative stress, chronic inflammation, apoptosis, and DNA damage, resulting in various adverse health effects, including cancer, cardiovascular disease, neurodegenerative conditions, and immune dysfunction. With recent advances in miRNA-based therapies, such as miRNA mimics and antagomiRs, there is growing potential for developing interventions that mitigate toxicant-induced cellular damage. This chapter seeks to explore the critical role that miRNAs play in mediating gene expression in response to toxicants (including transcriptional, posttranscriptional, and posttranslational mechanisms), focusing on how these small regulatory molecules contribute to cellular defense mechanisms and how their dysregulation can lead to adverse health outcomes. Key cellular responses, such as the DNA Damage Response (DDR) and p53 activation, work to maintain genomic stability by managing DNA repair, cell cycle control, and apoptosis.MiRNA profiling is an essential technique in miRNA biology, required to understand the role of individual miRNAs and their target genes. Quantitative polymerase chain reaction (qPCR) is a widely used method for miRNA profiling due to its high sensitivity and specificity. This protocol outlines a step-by-step approach for miRNA quantification using qPCR, focusing on the copy number assay and relative quantification methods. Through this exploration, this chapter aims to provide a comprehensive overview of the role of miRNAs in toxicology, highlighting their potential as both biomarkers and therapeutic targets in the context of environmental health and a protocol for quantification of miRNAs in different types of samples.