RNA profiling technologies. Several technology platforms are available for measuring RNA abundance on large scales. Microarray technologies rely on dense arrays of oligonucleotide probes used to capture complementary sequences present in biological samples at various concentrations. Following extraction, RNA is used as a template and amplified in a labeling reaction. The labeled material captured by the microarray is imaged and relative abundance determined based on the strength of the signal produced by the fluorochromes that serve as reporters in this assay. The Nanostring technology measures RNA abundance at the single molecule level. RNA serves as starting material for this assay, which does not involve the use of enzymes for amplification or labeling. Capture and reporter probes form complexes in solution with RNA molecules. These complexes are captured on a solid surface and imaged. Molecule counts are generated based on the number of reporter probes detected on the image. The reporter consists of a string of seven fluorochromes, with four different colors available to fill each position. Up to 500 different transcripts can be detected in a single reaction on this platform. For RNA sequencing (RNA-seq) the starting RNA population must first be converted into a library of cDNA fragments. High throughput sequencing of such fragments yields short sequences or reads that are typically 30 to 400 bp in length. For a given sample tens of millions of such sequences will then be uniquely mapped against a reference genome. The density of coverage for a given gene determines its relative level of expression. Similarities and differences between these technology platforms should be noted. For instance, microarrays and Nanostring technologies rely on oligonucleotide probes to capture complementary target sequences. Nanostring and RNA-seq technologies measure abundance at the single molecule level, with results expressed as molecule counts and sequence coverage, respectively. Microarray and RNA-seq technologies require extensive sample processing, which include amplification steps. dsDNA, double-stranded DNA.
Chaussabel et al. BMC Biology 2010 8:84 doi:10.1186/1741-7007-8-84