Essentials of transcriptomic methods: Navigating through RNA sequencing and beyond

RNA sequencing (RNA-seq) methodologies have evolved rapidly, offering powerful tools to study gene expression, transcriptome dynamics, and molecular mechanisms in various biological contexts. However, the complexity of these approaches poses challenges in data interpretation, sensitivity, and applicability. This chapter provides a comprehensive overview of RNA-seq methodologies, highlighting their advantages, limitations, and applications, particularly in cardiovascular research. Bulk RNA sequencing enables high-throughput gene expression profiling but lacks the resolution to capture cellular heterogeneity and spatial context. Direct RNA sequencing preserves native RNA modifications, offering insights into post-transcriptional regulation, though it remains technically challenging. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) bridge these gaps by resolving transcriptomic complexity at the cellular level and within tissue architecture, providing crucial insights into disease mechanisms such as atherosclerosis. By summarizing the strengths and limitations of these methodologies, this chapter aims to guide researchers in selecting the most suitable transcriptomic approach for their studies, ultimately advancing precision medicine and biomarker discovery in cardiovascular disease.