miRNA expression (by massive sequencing) - 2008
| Author: | S. Debernardi |
|---|---|
| Submitted: | Monday 12th of April 2010 01:59:29 PM |
| Submitted by: | egf |
| Educational levels: | expert, qc2, qc3 |
Topics
- Clinical/medical genetics
- Clinical/medical genetics > Patient related
- Clinical/medical genetics > Patient related > Patient/family related
- Clinical/medical genetics > Patient related > Patient/family related > Patient management
- Clinical/medical genetics > Patient related > Patient/family related > Patient management > Diagnosis of patient disease
- Clinical/medical genetics > Patient related > Patient/family related > Patient management > Diagnosis of patient disease > Laboratory investigation
- Clinical/medical genetics > Patient related > Patient/family related > Patient management > Diagnosis of patient disease > Laboratory investigation > Cytogenetics
- Molecular genetics
- Molecular genetics > Techniques
- Molecular genetics > Techniques > Molecular techniques
- Molecular genetics > Techniques > Molecular techniques > Sequencing
- Molecular genetics > Studies of RNA
- Molecular genetics > Studies of RNA > Non-coding RNA (miRNA/siRNA)
Abstract
It has been estimated that only 1.2 % of the human genome encodes proteins, however 60-70% of the mammalian genome is transcribed on one or both strands into non-protein coding RNAs 1. The untranslated fraction includes, among other small non-coding (snc)RNAs, the broad class of evolutionarily conserved molecules called miRNAs 2. Since Lee R.C. and colleagues 3 discovered the first gene of this family, lin-4 in C. elegans, several hundreds of miRNAs have been discovered in plants, animals and viruses. The miRBase database, a searchable online repository for published miRNA sequences, currently contains 8619 miRNAs from 58 species (http://microrna.sanger.ac.uk/ 4) and many more are predicted by computational analysis. MiRNAs have been shown to play key roles in a number of regulatory functions, including modulation of haematopoiesis and cell differentiation in mammals. They are transcribed from the genome as long primary molecules (pri-miRNAs), which are processed by the Drosha endonuclease to liberate a 60–70 nucleotides stem loop (or hairpin) precursor (pre-miRNA). The pre-miRNA is subsequently cleaved by the Dicer enzyme into the ~22 nucleotides mature miRNA. MiRNAs modulate gene expression through complementarity-mediated binding to the 3’ untranslated region (UTR) of target messenger RNAs (mRNAs), resulting in the repression of translation 5 or in the cleavage of the target transcript 6. On average, 200 genes are regulated by a single miRNA 7. Target genes can carry the recognition sequence for several miRNAs thus increasing the level of complexity. The binding of miRNAs to their target genes can be affected by RNA editing, for example adenosine (A) to inosine (I) RNA conversion 8 has been reported in a large fraction of the human transcriptome, including the primary transcripts of miRNAs 9,10. Terminal addition of A and uridine (U) nucleotides at the 3’ end of the miRNAs as well as internal nucleotide substitutions have also been reported 11. However, the extension and importance of miRNA editing remain largely unknown. The determination of genome-wide expression patterns for mature miRNAs is essential in the understanding of their function and in the study of disease biology. A number of DNA oligonucleotide arrays have been developed for miRNA expression studies both in normal human and mouse tissues 12,13 as well as in neoplastic human cells 14-16. Advances in microarray technologies, such as the introduction of locked nucleic acid probes 17 or the development of a bead-based, flow cytometric technique for obtaining miRNA expression profiles 18, have reduced the possibility of cross-hybridisation with closely related species and also with the precursor RNA molecules. An alternative strategy is based on real-time quantitative PCR by TaqMan assay designed to amplify only from the mature miRNA 19. The methods has been used in miRNA expression studies in human cancer 20,21 and offers the advantage of requiring a low amount of starting material. However previous experiments were limited to only a fraction of the miRNAs currently reported in the miRBase database and required prior knowledge of the miRNAs to be investigated. Newly developed high-throughput clonal sequencing technology 22,23 represents an unprecedented opportunity to uncover novel aspects of miRNA biogenesis, processing, and function. It offers the advantage over traditional sequencing technologies used for studying miRNA 11,24 of being less time consuming, relatively cheaper, and to allows the global unbiased determination of miRNA and other sncRNA expression patterns, including the discovery of novel species and the characterisation of enzymatic editing. It has been successfully applied for the investigation of sncRNAs among other species in human 25,26, mouse 27, C. elegans 28, and chicken 29. An overview of the most advanced high-throughput technologies for miRNA expression profiling will be presented here with examples of applications in human cancer studies.
Citation
S. Debernardi. miRNA expression (by massive sequencing) - 2008. EUROGENE portal. April 2010. online: http://eurogene.open.ac.uk/content/mirna-expression-massive-sequencing-2008
Keywords
Analysis of the entire coding region: Sequence analysis, antigen binding fragment, association, base, cell, centimorgan, correlation, cytoplasm, deoxyribonucleic acid, embryo, embryonic stem cell, expression, gene, gene expression, genome, high throughput, karyotype, library, ligase, marker, messenger rna, microarray, microrna, nucleus, oligonucleotide, open reading frame, polymerase chain reaction, primer, protein, reverse transriptase pcr, ribonucleic acid, selection, sequence, specie, transcription, translation, untranslated region, variationTerms of use
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