RNA sequencing (RNA-Seq) is a technique that uses next-generation sequencing to analyze the complete set of RNA transcripts in a cell or tissue sample. It provides a snapshot of gene expression, revealing which genes are active and at what levels.
How RNA-Seq Works
- RNA Extraction
Total RNA is extracted from the sample using methods similar to DNA isolation, but with additional steps to preserve the fragile RNA molecules. The RNA integrity is checked using gel electrophoresis or a bioanalyzer.
- mRNA Enrichment or rRNA Depletion
Since ribosomal RNA makes up most of the total RNA, it is removed to enrich for messenger RNA (mRNA). This is done using poly-T beads that capture the poly-A tails of mRNA, or by selectively depleting ribosomal RNA sequences.
- Library Preparation
The enriched mRNA is fragmented into small pieces and reverse transcribed into complementary DNA (cDNA) using random primers. Adapters are ligated to the cDNA fragments, and the library is amplified by PCR.
- Sequencing
The cDNA library is sequenced on an NGS platform, generating millions of short reads. Each read represents a short sequence from one end of a cDNA fragment.
- Data Analysis
The reads are aligned to a reference genome or transcriptome. The number of reads mapping to each gene is counted to quantify expression levels. Differential expression analysis identifies genes that are significantly up- or down-regulated between conditions.
- Applications
RNA-Seq is used to study gene expression changes in development, disease, drug treatment, and environmental responses. It can also detect alternative splicing, novel transcripts, and non-coding RNAs.
