RNA-Seq

Overview

Among different methods to evaluate gene expression, the high-throughput sequencing of RNA, or RNA-seq. is particularly attractive, as it can be performed and analyzed without relying on prior available genomic information. During RNA-seq, RNA isolated from samples of interest is used to generate a DNA library, which is then amplified and sequenced. Ultimately, RNA-seq can determine which genes are expressed, the levels of their expression, and the presence of any previously unknown transcripts.

Here, JoVE presents the basic principles behind RNA-seq. We then discuss the experimental and analytical steps of a general RNA-seq protocol. Finally, we examine how researchers are currently using RNA-seq, for example, to compare gene expression between different biological samples, or to characterize protein-RNA interactions.

Procedure

RNA sequencing, or RNA-seq, is a technique that can provide information on the sequence and quantity of every RNA expressed, known as the “transcriptome,” in a cell population. Unlike other expression profiling methods such as microarrays, which involve probing for known RNA sequences, RNA-seq can profile gene expression from organisms with un-sequenced genomes. Additionally, RNA-seq can accurately measure a larger range of transcript expression levels than microarrays, especially at very low or very high level