RNA sequencing

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The determination of the nucleotide sequence of the RNA is called RNA sequencing . For this, the RNA is translated into cDNA so that the method of DNA sequencing can be used. RNA-Seq reveals information about gene expression , such as how different alleles of a gene are expressed. In the case of RNA-Seq also the recognition of post-transcriptional modifications or identification of fusion genes .

overview

Various methods can be used after RNA purification . Basically, the technologies for researching gene expression can be divided into hybridization-based methods and sequence-based methods. Hybridization-based methods, such as B. Microarrays are relatively cheap, but these methods have some limitations, such as high background noise and a lower resolution ( dynamic range ). Sequence-based methods such as Sanger sequencing are very time-consuming and expensive, but have been further developed to SAGE and RT-PCR .

RNA-Seq is a modern sequence-based method and is based on next-generation sequencing (Engl. Next-generation sequencing ). RNA-Seq has clear advantages over the other methods. RNA-Seq helps to research complex transcriptomes and provides information about which exons come together in the messenger RNA . Low background noise, higher resolution and high reproduction rates in technical as well as biological replicas are clear advantages of RNA-Seq. However, "next-generation sequencing" techniques are comparatively expensive.

Biological background

The cell only uses part of its genes . This includes the household genes and the genes of the specialized cell. For example, muscle cells have mechanical properties and blood cells can carry oxygen. All cells have identical genes, but differ in their gene expression . Gene expression is the synthesis of proteins from DNA . The gene expression analysis or transcriptome analysis measures which genes are switched on or off. When a gene is switched on, parts of the gene are transferred into the mRNA . Methods of gene expression analysis, such as that of RNA-Seq, measure the concentration of the mRNA in various experimental conditions (e.g. with / without drugs). The gene expression analysis thus follows the question of how the mRNA concentration behaves as a result of drugs, in different development stages of the cell, in a healthy or diseased state.

The RNA sequence can be used to better understand the mechanism of alternative splicing and fusion genes. Alternative splicing is the process in which the pre-RNA is converted into different mRNAs and thus also different proteins. Fusion genes are hybrid genes from two previously separated genes, united in one gene. Fusion genes arise through translocation , interstitial deletion or chromosomal inversion .

Individual evidence

  1. ^ A b Zhong Wang, Mark Gerstein, Michael Snyder: RNA-Seq: a revolutionary tool for transcriptomics . In: Nature Reviews Genetics . 10, No. 1, January 2009, pp. 57-63. doi : 10.1038 / nrg2484 . PMID 19015660 . PMC 2949280 (free full text).
  2. ↑ Thomas E. Royce, Joel S. Rozowsky, Mark B. Gerstein: Toward a universal microarray: prediction of gene expression through nearest-neighbor probe sequence identification. . In: Nucleic Acids Res . 35, No. 15, 2007, p. E99. doi : 10.1093 / nar / gkm549 . PMID 17686789 . PMC PMC1976448 (free full text).
  3. ↑ Michał J. Okoniewski, Crispin J. Miller: Hybridization interactions between probesets in short oligo microarrays lead to spurious correlations. . In: BMC Bioinformatics . 7, 2006, p. 276. doi : 10.1186 / 1471-2105-7-276 . PMID 16749918 . PMC PMC1513401 (free full text).
  4. ↑ Trapnell C, Pachter L, Salzberg SL: Top Hat: discovering splice junctions with RNA-Seq. . In: Bioinformatics . 25, No. 9, 2009, pp. 1105-1111. doi : 10.1093 / bioinformatics / btp120 . PMID 19289445 . PMC PMC2672628 (free full text).
  5. ↑ Teixeira MR: Recurrent fusion oncogenes in carcinomas. . In: Crit Rev Oncog . 12, No. 3-4, 2006, pp. 257-271. PMID 17425505 .