The completion of the draft and complete human genome has revealed that there are only around 20,000 genes encoding proteins. Nonetheless, these genes can generate eight times more RNA transcript isoforms, while this number is still growing with the accumulation of high-throughput RNA sequencing (RNA-seq) data. In general, over 90% of genes generate various RNA isoforms emerging from variations at the 5′ and 3′ ends, as well as different exon combinations, known as alternative transcription start site (TSS), alternative polyadenylation (APA), and alternative splicing (AS). In this chapter, our focus will be on introducing the significance of these three types of isoform variations in gene regulation and their underlying molecular mechanisms. Additionally, we will highlight the historical, current, and prospective technological advancements in elucidating isoform regulations, from both the computational side such as deep-learning-based artificial intelligence, and the experimental aspect such as the long-read third-generation sequencing (TGS).