- RNA-sequencing (RNA-seq) is a powerful and widely used technique for studying the transcriptome — the complete set of RNA transcripts produced by the genome under specific circumstances or in a particular cell type.
- It allows researchers to capture a snapshot of gene expression and gain insights into the dynamic changes in transcription across different biological conditions.
- Unlike earlier methods such as microarrays, RNA-seq does not require prior knowledge of the transcript sequences, making it particularly valuable for discovering novel transcripts, splice variants, and gene fusions.
- The RNA-seq process begins with the extraction of total RNA from a biological sample, which is then typically enriched for messenger RNA (mRNA) or depleted of ribosomal RNA (rRNA) to focus on coding and non-coding RNAs of interest. The RNA is then fragmented into smaller pieces, converted into complementary DNA (cDNA) using reverse transcription, and ligated with sequencing adapters. This library is subsequently amplified and subjected to high-throughput sequencing using platforms such as Illumina, Oxford Nanopore, or PacBio. The resulting reads are aligned to a reference genome or assembled de novo, allowing quantification of transcript abundance and identification of transcript structure.
- RNA-seq enables comprehensive analysis of gene expression levels, alternative splicing, post-transcriptional modifications, non-coding RNAs, and allele-specific expression. It is particularly useful in comparing transcriptomes between healthy and diseased tissues, revealing differentially expressed genes that may be involved in disease mechanisms or serve as potential biomarkers.
- Additionally, single-cell RNA-seq (scRNA-seq) has emerged as a transformative extension of the method, allowing gene expression profiling at the level of individual cells, thus uncovering cellular heterogeneity within complex tissues.
- Overall, RNA-sequencing has revolutionized molecular biology and biomedical research by providing a detailed and unbiased view of the transcriptome, with applications ranging from basic science to clinical diagnostics and therapeutic development.
