MIRNAS ARRAYS: DATABASES AND PLATFORMS UBio Training Courses Gonzalo Gómez//[email protected].
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Transcript of MIRNAS ARRAYS: DATABASES AND PLATFORMS UBio Training Courses Gonzalo Gómez//[email protected].
MIRNAS ARRAYS: DATABASES AND PLATFORMSUBio Training Courses
Gonzalo Gómez//[email protected]
miRNAs
Firstly detected in C. elegans (V. Ambros, 1993)
Mapping to non-coding regions (introns)
Pri-miRNA processed by Drosha
DICER removes the structural loop
mature miRNA: ssRNA, 22 nucleotides
miRNA-RISK complex: mRNAs post-transcriptional inhibition
33% of human genes are supposed to be regulated by miRNAs.
miRNAs
Biological role
• Brain development (miR-430)
• Nervous system development(miR-273)
• Pancreatic Langerhans islands development (miR-375)
• Adipocytes development (miR-143)
• Heart development(miR-1)
• Inmune response (miR-223, cluster miR-17~92, miR-146a,miR-155…)
• Apoptosis (miR-14)
miRNAs and cancerOncomiRs
Esquela-Kerscher & Slack. Nature Reviews Cancer. 2006.
ProliferationInvasionAngiogenesisCell death
Oncogene miRNA
Proliferation Invasion AngiogenesisCell death
Tumor suppressor miRNA
Tumor formation
Upregulation
Downregulation
miRNAs
Target sites
Bartel D. Cell 2009.
miRNAs seed region: 5’, nucleotides 2-7(8)
Most gene targets of a given miRNA have only a single 7 nt matching to that miRNA seed region.
7-8 nt hundreds of target predictions for each miRNA family (~300 conserved targets per miRNA family in vertebrates)
High rate of false positives in predictions.
miRNAs
Target Prediction Algorithms
PREDICTION CRITERIA
• miR seed-target complete base-
pairing
• Interspecies conservation
• Number of binding sites for a given 3’UTR
in a particular gene
• Free energy for the miR-target duplex
• Binding site accessibility
• miRNA- target secondary structure
Other prediction algorithms…Bartel D. Cell 2009.
More target prediction tools: http://en.wikipedia.org/wiki/
List_of_RNA_structure_prediction_software#Inter_molecular_interactions:_MicroRNA:UTR
Version Date miRNAs
1.0 12/02 218 1.1 01/03 262 1.2 04/03 295 1.3 05/03 332 1.4 07/03 345 2.0 07/03 506 2.1 09/03 558 2.2 11/03 593 3.0 01/04 719 3.1 04/04 899 4.0 07/04 1185 5.0 09/04 1345 5.1 12/04 1420 6.0 04/05 1650 7.0 06/05 2909 7.1 10/05 3424 8.0 02/06 3518 8.1 05/06 3963 8.2 07/06 4039 9.0 10/06 4361 9.1 02/07 4449 9.2 05/07 4584 10.0 08/07 5071 10.1 12/07 5395 11.0 04/08 6396 12.0 09/08 8619 13.0 03/09 9539 14.0 09/09 10833
H. Sapiens ~695 miRNAsM. musculus: ~488 miRNAs
miRNAs
Databases
http://www.mirbase.org/
miRBase
miRNAs
a) mir: immature sequence (hairpin). E.g. hsa-mir-203
b) miR: mature miRNA sequence. E.g. hsa-miR-203
- a/b: paralog miRs, difer in 1-2 nucleotides.
E.g. hsa-miR-9a, hsa-miR-9b
- 1-2: Identical miRs, different hairpin.
Ej. hsa-miR-19b-1, hsa-miR-19b-2
- 5p-3p: mature miR generated from precursor 5´ (or 3) sequence.
E.g. hsa-miR-17-5p
- *: Minor transcript complementary to mature miR.
E.g. hsa-miR-33a*
miRNA nomenclature (miRBase)
TarBase http://microrna.gr/tarbase
miRNAs
Databases
The database of experimentally supported targets: a functional update of TarBase. Papadopoulos GL, Reczko M, Simossis VA, Sethupathy P, Hatzigeorgiou AG., Nucleic Acids Res. 2009 Jan;37(Database issue):D155-8.
Contains only those miRNA-target relationships experimentally validated
miRNAs and disease
http://www.mir2disease.org/
Cancer (Calin and Croce 2006; Ura et al 2008; Stamatopoulos et al 2009…)Cardiovascular disease (Latronico et al. 2007; van Rooij and Olson 2007)
Schizophrenia (Hansen, et al. 2007; Perkins et al. 2007)Renal misfunction (Williams 2007)
Tourette syndrome (Esau and Monia 2007)Psoriasis (Sonkoly et al. 2007)
Muscle disorders (Eisenberg et al. 2007),X fragile syndrome (Fiore and Schratt 2007)
Policitemia vera (Bruchova et al. 2007)Diabetes (Williams 2007)
Chronic hepatitis (Murakami et al. 2006)AIDS (Hariharan etal. 2005)
Obesity (Weiler et al. 2006, Lovis et al. 2008, Xie et al. 2009).
http://cmbi.bjmu.edu.cn/hmdd
Databases
miRNAsDetection Methods
miRNA microarrays
Commercial platforms
Human, rat, mouse
Human, rat, mouse, dog, chimpanzee, etc
Human, multispecie
Human, rat, mouse
miRNA microarrays
Commercial platforms
Agilent recommendations.-No normalization-Normalization 75th percentile (75th value = 1)
Additional G-C pair in the probe-target interaction region stabilizes targeted miRNAs relative to homologous RNAs. Additionally, all probes contain a 5' hairpin (blue), abutting the probe-target region, to increase target and size miRNA specificity
miRNA microarrays
Commercial platforms
Background correction = normexpNormalization = quantiles
RG_normexp <- backgroundCorrect(RGfilt_0, method = "normexp", offset=50)MA_norm <- normalizeBetweenArrays(RG_normexp$G, method="quantile")MA_lognorm<-log2(MA_norm)
RG_normexp <- backgroundCorrect(RGfilt_0, method = "normexp", offset=50)MA_norm <- normalizeBetweenArrays(RG_normexp$G, method="quantile")MA_lognorm<-log2(MA_norm)
LNA = Locked Nucleic Acid
LNA is chemical modification.Ribose ring is "locked" with a methylene bridgeconnecting the 2’-O atom and the 4’-C atom.
LNA makes DNA-miR pairs more stable (higher Tm)when perfect match and 1mismatch hybridizations occurs.
Preprocessing scripts provided by Exiqon
Differentially expressed miRNAs between classes
T test, SAM, limma
FWERFDR
METHOD
Pvalueadjustment
FWER: Type I Family Wise Error RateFDR: False Discovery Rate
OK!pvalue
20 normalized arrays600 miRNAs 2 classes (healthy y tumor)
miRNA microarrays
Differential expression analysis
GEPASAsteriasSAM tools…
THANKS!
http://bioinfo.cnio.es/
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