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: Cy3 vs Cy5)

3. 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 - 90% amplification - Single product in most PCRs 10,0 00 3,00 0 1,000 10,0 00 3,00 0 1,000 10,00 0 3,000 10,0 00 3,00 0 1,000 21,226 5,148 2,000 21,226 5,148 2,000 21,226 5,148 2,000 21,226 5,148 2,000

4. Operon D. melanogaster Array - 10 A. thaliana oligos (TIGR spikes) - each printed 4 times by pin = 640 spots - 12 D. melanogaster oligos - each printed 17 times = 204 spots 16416 spots - 12 Randomly Generated Negative Controls – printed several times = 188 spots - 352 Empty spots - 449 Buffer spots 14593 70mer probes representing 13664 genes and 17899 transcripts POSITIVE CONTROLS NEGATIVE CONTROLS

6. Hybridization of Chips mutant flies (ash2) wild-type flies Fluorescent Labelling RNA Extraction mRNA Cy5 test sampleCy3 control sample Hybridize Slide

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

8. Operon D. melanogaster Array - 10 A. thaliana oligos (TIGR spikes) - each printed 4 times by pin = 640 spots - 12 D. melanogaster oligos - each printed 17 times = 204 spots 16416 spots - 12 Randomly Generated Negative Controls – printed several times = 188 spots - 352 Empty spots - 449 Buffer spots 14593 70mer probes representing 13664 genes and 17899 transcripts POSITIVE CONTROLS NEGATIVE CONTROLS (hybridized with aRNA ISOash2 I1 vs ISO)

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

10. TIGR spike-in Mix 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: We can use the spikes to assess quality of experiment and analysis For Amplification experiments we use the spikes diluted 1:500

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

12. “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

13. Adjusting Ratios - 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. - A Ratio measures how much sample cDNA over control cDNA we have of a given gene. This is: Ratio = Intensity sample / Intensity control

14. - We expect: - few genes upregulated - few genes downregulated - most genes unchanged (log 2 Ratio = 0) -Therefore: - a Normal distribution - with mean (all log 2 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)

15. Multiple Experiment Comparison

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

17. 1 MEV file for experiment (total=5) 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

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

19. 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?

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