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Transcriptome analysis: RNA-seq

RNA-seq is an high throughput technology used to identify the presence and the quantity of RNA in a biological sample in a given moment. It provides far more precise measurement of levels of transcripts and their isoforms than other methods, allowing researchers to better analyze the transcriptome: the the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physiological/pathological condition. The key aims of transcriptomics are: 

• to catalogue all species of transcript, including mRNAs, non-coding RNAs and small RNAs; 
• to determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications; 
• to quantify the changing expression levels of each transcript during development and under different conditions

In general, a population of RNA is converted into a cDNA library with adaptors attached at one or both ends. Then, each molecule is sequenced to obtain informations from one end (single-end sequencing) or both ends (pair-end sequencing). The sequenced reads (generally 30-400 bp long, depending on the used machinery) are then aligned on a reference genome or transcriptome or de novo assembled to produce a genome-scale transcription map (also expression levels of different genes might be reported).

(Danilo Lombardi)