Diapositiva 1 - Docenti.unina
annuncio pubblicitario
Un solo genoma con una molteplicità impressionante di varianti di espressione Corteccia cerebrale Fegato Muscolo liscio Il Trascrittoma è l’insieme completo dei trascritti e dei loro livelli di espressione in un dato tipo cellulare ed un determinato stato fisiologico. Transcriptome studies a)Mappare “sequence reads” onto a reference genome. b) De novo transcriptome assembly uses software to infer transcripts directly from short sequence reads. c) Differential expression •A eukaryotic cell contains a total mass of ∼100 000 mRNAs with ∼15.000–30.000 distinct mRNAs. Prevalence ranging from one to several thousands. •About 50% of the transcript population is made up of a relatively small number (some hundreds) of abundant transcripts, representing only 1% of the different mRNA species. •The other 50 % half contains the ‘rare’ mRNAs. •The difficulty of fishing out a gene responsible for a specialized function in a certain biological program often originates from the fact that the gene is expressed at low levels whereas the bulk of a cell’s mRNA is made up of highly abundant transcripts. Analisi di espressione differenziale Screening for differentially expressed genes is one of the most straightforward approaches to unravel the molecular basis of a biological system Le cDNAteche sono uno strumento prezioso per gli studi di espressione genica Conversione dei trascritti in cDNA Selection /Isolation of specific messenger RNA Identification of novel messenger RNA with degenerated oligo/probes Studies on differentially expressed messenger RNA Differential Hybridization Technique Differential hybridization o ibridazione sottrattiva driver cDNAteca Driver Marcata con biotina Biotina: cofattore Avidina ne è ricco il bianco d’uovo essa lega “avidamente la Biotina” e ne previene l’assorbimento intestinale;. tester Differential Dysplay Technique RT-PCR to increase mRNA abundance AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT AAAAAAAA TTTTTTTTT RNAsi Primer sequenza specifico TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT Primer hybridization high stringency TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT PCR Oligo dTTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT PCR Oligo dTTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT PCR TTTTTTTTT Oligo dTTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTTTA3’OH TTTTTTTTTTT TTTTTTTTTTG TTTTTTTTTTC TTTTTTTTTTCG TTTTTTTTTTCC Differentially expressed gene Misure di espressione genica: 1980 Northern Blot/Dot Blot Misure di espressione genica: 1990 Differential dysplay e tecniche sottrattive /PCR 1995 Arrays/ SAGE/RealTime PCR wide”) (analisi “genome The complexity of transcriptome http://neomorph.salk.edu/human_methylo me/data.html DNA Transcription 20.929 Protein coding RNA Translation 23.218 Non-coding RNA No Translation Protein More than 70% of the genome is transcribed and 50% is non-coding RNA Complex splicing pattern La complessità del trascrittoma Alternative promoters Complex splicing patterns Messenger RNA Tissue-specificity of promoters Corteccia somato-sensitiva Ippocampo Corteccia visiva Cervelletto Differentiation stage-specificity of promoters XM (mRNA) XR (non-coding RNA) transcript from genome annotation NM NR XP( protein) NCBI predicted NP from RNA sequencing and may differ from XP files Fantom 5 project: Alternative Transcription Starting Sites (TSS) Proteina fibrillare acida della glia Actin B
