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MICROARRAYS D’EXPRESSIÓ ESTUDI DE REGULADORS DE LA TRANSCRIPCIÓ DE LA FAMILIA trxG

MICROARRAYS D’EXPRESSIÓ ESTUDI DE REGULADORS DE LA TRANSCRIPCIÓ DE LA FAMILIA trxG. M. Corominas : mcorominas@ub.edu. Spotted microarrays rely on delivery technologies to place biologic material (purified cDNA, oligonucleotides) onto allocated locations of the chip.

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MICROARRAYS D’EXPRESSIÓ ESTUDI DE REGULADORS DE LA TRANSCRIPCIÓ DE LA FAMILIA trxG

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  1. MICROARRAYS D’EXPRESSIÓ ESTUDI DE REGULADORS DE LA TRANSCRIPCIÓ DE LA FAMILIA trxG M. Corominas: mcorominas@ub.edu

  2. Spotted microarrays rely on delivery technologies to place biologic material (purified cDNA, oligonucleotides) onto allocated locations of the chip. (competitive hybridization: Cy3vsCy5)

  3. - 90% amplification - Single product in most PCRs 10,000 3,000 21,226 5,148 2,000 1,000 21,226 5,148 2,000 10,000 3,000 1,000 10,000 3,000 21,226 5,148 2,000 Direct PCR from Bacterial Growth 1,000 21,226 5,148 2,000 10,000 3,000 Analysis of PCR results by electrophoresis Spotting on slide 1,000 Production of cDNA chips 17 plates from the Berkeley Drosophila Gene Collection with 384 wells (clones) each. Aprox. 5000 genes in total

  4. 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

  5. RNA Extraction mRNA mRNA Cy5 test sample Cy3 control sample Hybridize Slide Hybridization of Chips mutant flies (ash2) wild-type flies Fluorescent Labelling

  6. 532 nm 635nm -Integrate Data -Filter Data -Adjust dye bias -Calculate Ratios -Adjust Data -Set Thresholds Scanning of Chips Scan Slide fluorescent intensities for each cDNA, spot or gene fluorescent intensities for each cDNA, spot or gene GenePix

  7. 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)

  8. Amplification Test: totalRNA vs aRNA log2ratios Correlation coef = 0.94

  9. 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: For Amplification experiments we use the spikes diluted 1:500

  10. TIGR spikes MA plot from an experiment with total RNA Experimental procedure and analysis seems good (spikes fall where expected)

  11. “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

  12. 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 intensitiesmore  independent of absolute magnitude. • Gives a more realistic sense of variation.

  13. Therefore: • a Normal distribution • with mean (all log2 Ratio ) = 0 • We expect: • few genes upregulated • few genes downregulated • most genes unchanged (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)

  14. Norm log Ratio of Medians Experiment 1 Experiment 3 Experiment 2 Experiment 4 7 6 5 4 % Genes in Class 3 2 1 0 4 -7 0.7 1.8 2.9 5.1 6.2 -1.5 -5.9 -4.8 -3.7 -2.6 -0.4 log Ratio of Medians Class Multiple Experiment Comparison

  15. 2 TIFF images (Cy3 & Cy5) GAL file (gene matrix) Input GenePix Pro 4.0 Image analysis ANALYSIS LAYOUT Output 1 GPR file for experiment Input TIGR Express Converter 1.4.1 Output 1 MEV file for experiment

  16. 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

  17. Controls and Quality assesment - Sequencing of some clones from the Collection plates - RT-PCR of some genes in a semiquantitative way - in situ hybridization - Western Blot - inmunolocalization - Northern Blot - Clonal Analysis

  18. Classification according to GO (Gene Ontology) • Gene Ontology is a “controlled vocabulary that can be • applied to all eukaryotes “. Each gene product is classi- • fied in one or more categories. • Is distribution of missexpressed genes significantly • different from the one of our initial set of genes? • maybe trxG genes act predominantly upon a group • of genes of similar function or pathway?

  19. Departament de Genètica: Florenci Serras Montserrat Corominas Isabel Almudí Mireia Angulo Sergi Beltran Cristina Pallarès Miguel Pignatelli Adrià Punset Marta Sesé CRG-UPF-IMIM Roderic Guigó Enrique Blanco Plataforma de Transcriptòmica Parc Científic- SCT-UB Lídia Sevilla

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