RNA sequencing (RNA-Seq) has transformed how researchers study transcriptomes. It provides a quantitative and qualitative view of all RNA molecules within a cell at a given time. Unlike microarrays, RNA-Seq does not require prior knowledge of the genome, enabling unbiased discovery of novel transcripts, splice variants, and gene fusions.
What is RNA-Seq?
RNA-Seq is a next-generation sequencing (NGS) technology that sequences complementary DNA (cDNA) derived from RNA molecules. It allows researchers to capture the dynamic nature of transcriptomes, offering high-resolution insights into gene expression patterns.
Steps Involved in RNA-Seq
1. RNA Extraction: Total RNA is isolated from the sample.
2. Library Preparation: RNA is converted into cDNA and adapters are ligated for sequencing.
3. Sequencing: Using NGS platforms such as Illumina, libraries are sequenced to generate short reads.
4. Data Analysis: Reads are aligned to a reference genome or transcriptome. Expression quantification and differential expression analysis are performed.
Applications of RNA-Seq
RNA-Seq is widely used in:
- Gene expression profiling
- Transcriptome annotation
- Alternative splicing analysis
- Fusion gene detection
- Single-cell transcriptomics
Conclusion
RNA-Seq is an essential tool in functional genomics and systems biology. Its ability to reveal the complexity of gene expression and transcript diversity has made it invaluable for researchers in medicine, plant science, microbiology, and beyond.