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RNA-Seq: a revolutionary tool for transcriptomics


RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes.

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Figure 1: A typical RNA-Seq experiment.
Figure 2: Quantifying expression levels: RNA-Seq and microarray compared.
Figure 3: DNA library preparation: RNA fragmentation and DNA fragmentation compared.
Figure 4: Poly(A) tags from RNA-Seq.
Figure 5: Coverage versus depth.

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We thank D. Raha for many valuable comments.

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Correspondence to Michael Snyder.

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Cap analysis of gene expression

(CAGE). Similar to SAGE, except that 5′-end information of the transcript is analysed instead of 3′-end information.


A group of sequences representing overlapping regions from a genome or transcriptome.

dsRNA editing

Site-specific modification of a pre-mRNA by dsRNA-specific enzymes that leads to the production of variant mRNA from the same gene.

Genomic tiling microarray

A DNA microarray that uses a set of overlapping oligonucleotide probes that represent a subset of or the whole genome at very high resolution.

Massively parallel signature sequencing

(MPSS). A gene expression quantification method that determines 17–20-bp 'signatures' from the ends of a cDNA molecule using multiple cycles of enzymatic cleavage and ligation.


(miRNA). Small RNA molecules that are processed from small hairpin RNA (shRNA) precursors that are produced from miRNA genes. miRNAs are 21–23 nucleotides in length and through the RNA-induced silencing complex they target and silence mRNAs containing imperfectly complementary sequence.

Piwi-interacting RNAs

(piRNA). Small RNA species that are processed from single-stranded precursor RNAs. They are 25–35 nucleotides in length and form complexes with the piwi protein. piRNAs are probably involved in transposon silencing and stem-cell function.

Quantitative PCR

(qPCR). An application of PCR to determine the quantity of DNA or RNA in a sample. The measurements are often made in real time and the method is also called real-time PCR.

Sequencing depth

The total number of all the sequences reads or base pairs represented in a single sequencing experiment or series of experiments.

Serial analysis of gene expression

(SAGE). A method that uses short 14–20-bp sequence tags from the 3′ ends of transcripts to measure gene expression levels.

Short interfering RNA

(siRNA). RNA molecules that are 21–23 nucleotides long and that are processed from long double-stranded RNAs; they are functional components of the RNAi-induced silencing complex. siRNAs typically target and silence mRNAs by binding perfectly complementary sequences in the mRNA and causing their degradation and/or translation inhibition.

Spike-in RNA

A few species of RNA with known sequence and quantity that are added as internal controls in RNA-Seq experiments.

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Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10, 57–63 (2009).

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