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MICROARRAYS D’EXPRESSIÓ ESTUDI DEL FACTOR DE TRANSCRIPCIÓ ASH2 M. Corominas: [email protected] Spotted array experiment 1. Prepare sample. Test 4. Print microarray. Reference 2. Label with fluorescent dyes. 5. Hybridize to microarray. 3. Combine cDNAs. 6. Scan. Spotted microarrays rely on delivery technologies to place biologic material (purified cDNA, oligonucleotides) onto allocated locations of the chip. (competitive hybridization: Cy3 vs Cy5) Drosophila melanogaster Wolpert (2001) ash2 - member of a trithorax group Belongs to multiprotein chromatin remodeling complexes -Polycomb (PcG) : transcriptional repression -trithorax (trxG) : transcripcional activation Transcriptional Regulator TYPES OF MICROARRAYS 1) From full length cDNA Plates from the Berkeley Drosophila Gene Collection with 384 wells (clones) each: DGC1.0 and 2.0 Aprox. 12000 genes in total Direct PCR from Bacterial Growth using vector-specific primers Analysis of PCR results by electrophoresis Spotting on slide TYPES OF MICROARRAYS 2) From 400 bp amplicons a) correspond to approximately 75% of genes predicted in release 3.1 (gene specific primers kindly donated by Incyte Genomics and Brian Oliver, NIH). b) based on a novel annotation of the fly genome. It contains 21376 gene- specific probes. Performed and available from Eurogentec. Carried out in collaboration (ZMBH, Univ. of Heidelberg; DKFZ, MPI Molecular Genetics Computational Molecular Biology, Germany) 3) From oligonucleotides a) INDAC project: International Drosophila Array Consortium www.indac.net 70 mer oligonucleotides designed towards the 3’ end of the genes (based on the 3.1 release) with specific algorithms and synthesized by Illumina. b) Qiagen/Operon oligo set 70 mer oligonucleotides representing 13,664 genes designed from release 3.1 already available in the Plataforma de Transcriptòmica Serveis Científico-Tècnics UB- PCB - RNA samples: - total RNA - polyA+ RNA - T7 polymerase amplified RNA - labeling method (competitive hybridization): - direct - indirect - positive and negative controls MIAME describes the Minimum Information About a Microarray Experiment that is needed to enable the interpretation of the results of the experiment unambiguously and potentially to reproduce the experiment. http://www.mged.org/Workgroups/MIAME/miame.html Production of cDNA chips 17 plates from the Berkeley Drosophila Gene Collection with 384 wells (clones) each. Aprox. 5000 genes in total Direct PCR from Bacterial Growth Analysis of PCR results by electrophoresis Spotting on slide Hybridization of Chips mutant flies (ash2) wild-type flies Trizol RNA Extraction & Poly A+ Purification mRNA mRNA Two-Step Fluorescent Labelling Cy5 test sample Cy3 control sample Hybridize Slide Scanning of Chips 532 nm Scan Slide fluorescent intensities for GenePix each cDNA, spot or gene -Integrate Data -Filter Data -Adjust dye bias 635nm fluorescent intensities for each cDNA, spot or gene -Calculate Ratios -Adjust Data -Set Thresholds “Bad” Spots Filtering - Is the process in which spots that don’t look right are discarded according to different criteria GenePix discards data according to internal filters like: x % pixels > Median Background intensity Convert Data 3.33 to further filter data. Spots were flagged as OK if: medianFx > mBx +/- XSD - Spots must pass filtering for both channels log (F Median - B) 17.8 17 16.2 15.4 14.6 13.8 13 12.2 11.4 10.6 9.8 9 8.2 7.4 6.6 5.8 5 Number in each class 80 60 40 20 0 log(F532Median-B532) log(F635Median-B635) 120 Distribution for Good spots at both wavelengths 100 Adjusting Ratios - A Ratio measures how much sample cDNA over control cDNA we have of a given gene. This is: Ratio = Intensity sample / Intensity control - Different measures for the ratios: - Ratio of Medians - Ratio of Means - Regression Ratio -Log (base 2) the ratios : •Makes variation of intensities and ratios of intensities more independent of absolute magnitude. •Gives a more realistic sense of variation. Multiple Experiment Comparison Modify data the same way in all experiments: - bad spots filtering methods - ratio (eg. Ratio of Medians) - adjust ratios: - mean centering - Normalization - main class centering - We expect: - few genes upregulated - few genes downregulated - most genes unchanged (log2 Ratio = 0) -Therefore: - a Normal distribution - with mean (all log2 Ratio ) = 0 -Draw distribution of Ratios and check mean: - if really not N: filter bad spots better try to Normalize (mean = 0; SD = 1) discard experiment - if close to N: adjust mean (product or sum) Normalize (0; 1) Multiple Experiment Comparison Norm log Ratio of Medians Experiment 3 Experiment 4 7 6 5 4 3 2 log Ratio of Medians Class 6.2 5.1 4 2.9 1.8 0.7 -0.4 -1.5 -2.6 -3.7 -4.8 0 -5.9 1 -7 % Genes in Class Experiment 1 Experiment 2 Set method to select up or downregulated genes - higly subjective method like fold-change (eg. two, three) - semi-statistical method like Mean ± xSD - statistical method like SAM: - missing values imputed using a K-nearest Neighbor - computes a statistic - set threshold for statistic (to call significant genes) - will give you a FDR - set fold-change threshold Results 5139 different genes with FBgn in total Mean Corr. Coef 0.88 4163 different genes with FBgn (SAM INPUT) SAM 2.5% FDR 1.75 Foldchange 95 140 Filtering of Bad Spots -If a gene is downregulated in the mutant (ash2I1): Ratio = F sample / F control <1 log2 Ratio <0, because log2 1=0 ash2 is in activation pathway - If a gene remains unchanged in the mutant (ash2I1): Ratio = F sample / F control = 1 log2 Ratio = 0, because log2 1=0 ash2 is not regulating this gene -If a gene is upregulated in the mutant (ash2I1): Ratio = F sample / F control > 1 log2 Ratio > 0, because log2 1=0 ash2 is in repression pathway Controls and Quality assesment - Sequencing of some clones from the Collection plates - RT-PCR of some genes in a semiquantitative way - Western Blot - in situ hybridization - Northern Blot - inmunolocalization - Clonal Analysis RT-PCR + = wt ASH2 - = ash2 Classification according to GO (Gene Ontology) - Gene Ontology is a “controlled vocabulary that can be applied to all eukaryotes “. Each gene product is classified in one or more categories. - Is distribution of missexpressed genes significantly different from the one of our initial set of genes? - maybe ash2 acts predominantly upon a group of genes of similar function or pathway 20.0 17.5 15.0 12.5 7.5 5.0 2.5 0.0 19 24 29 34 39 3 18S 28S Fluorescence 10.0 44 Time (seconds) 49 54 59 64 69 Operon D. melanogaster Array 16416 spots 14593 70mer probes representing 13664 genes and 17899 transcripts POSITIVE CONTROLS - 10 A. thaliana oligos (TIGR spikes) - each printed 4 times by pin = 640 spots - 12 D. melanogaster oligos - each printed 17 times = 204 spots NEGATIVE CONTROLS - 12 Randomly Generated Negative Controls – printed several times = 188 spots - 352 Empty spots - 449 Buffer spots (hybridized with aRNA ISOash2I1 vs ISO) ANALYSIS LAYOUT 2 TIFF images (Cy3 & Cy5) GAL file (gene matrix) Input GenePix Pro 4.0 Image analysis Output 1 GPR file for experiment Input TIGR Express Converter 1.4.1 Output 1 MEV file for experiment 1 MEV file for experiment (total=5) Input TIGR MIDAS - Each experiment analyzed independently - Background filter applied - Normalization applied: Lowess (LOC) for each experiment independently Input EXCEL & TIGR MEV - Spike-in, negative and positive control Check - MA Plots - Experiment Comparison (Scatter Plots) - Relevant Genes Finding TIGR spike-in Mix We can use the spikes to assess quality of experiment and analysis On chip: 10 A. thaliana oligos spotted 64 times each (4 times by pin) To add to labeling reaction: In vitro synthesized RNA from each gene at different proportions and quantities: GENE RCA Cab RbcL Ltp4 Ltp6 PRK TIM Nac RCP XCP Ratio 1 to 1 1 to 1 1 to 1 1 to 1 2 to 1 2 to 1 2 to 1 1 to 3 1 to 3 1 to 3 pg in 2 ul of: Mix A Mix B 5000 5000 2000 2000 500 5000 20 20 3000 1500 500 250 100 50 10 30 200 600 1000 3000 For Amplification experiments we use the spikes diluted 1:500 TIGR spikes MA plot from an experiment with total RNA Experimental procedure and analysis seems good (spikes fall where expected) DOO-016TIGR Spikes MA Plot 3 2 RCA (11) CAB (11) 1 rbcL (11) log2(Cy5*Cy3) LTP4 (11) XCP2 (13) RCP1 (13) 0 NAC1 (13) Ltp6 (21) PRKase (21) -1 TIM (21) 3 to 1 ratio 1 to 2 ratio -2 -3 27 32 37 log2(Cy5/Cy3) 42 47 Operon Arrays Insets ISO ash2I1 vs ISO L3 total RNA - 60ug indirectly labelled aRNA from L3 total RNA - 2 ug amplified to 70ug in 4h - 20ug of labelled aRNA TIGR Spikes Amplification Test: totalRNA vs aRNA log2ratios Correlation coef = 0.94 Biological Replicates REPLICATE 1 REPLICATE 2 35 35 30 30 25 25 20 Fluorescence 20 Fluorescence 15 15 10 10 5 5 0 0 24 29 34 39 44 18S 28S 18S 28S 19 49 54 59 64 69 19 Time (seconds) 29 34 39 44 49 54 59 64 69 Time (seconds) 12.5 10.0 10.0 7.5 7.5 Fluorescence 12.5 Fluorescence 24 5.0 2.5 5.0 2.5 0.0 0.0 19 24 29 34 39 44 Time (seconds) 49 54 59 64 69 19 24 29 34 39 44 Time (seconds) 49 54 59 64 69 Biological Replicates Microarray Insets REPLICATE 1 REPLICATE 2 Amplified TIGR spikes (diluted 1:100) together with probes Biological Replicates Replicate 1 vs Rplicate 2 log2ratios Correlation coef = 0.92