1. Microarray (Gene Expression) DNA microarrays is a technology that can be used to measure changes in expression levels or to detect SNiPs Microarrays differ in fabrication, workings, accuracy, efficiency, and cost Additional factors for microarray experiments are the experimental design and the methods of analyzing the data Historically, the technique evolved from southern blotting Think microscale for hybridization of many genes 1

2. Can be used to pinpoint all (most) the differences between gene expression between two different cell/tissue types……..in a single experiment. Senescing Leaf Green Leaf DNA microarray 2

3. Isolation of mRNA, which is relatively unstable and short lived Microarrays measures relative abundance of levels of gene expression mRNA directs the production of cellular proteins (although protein synthesis and activation are not regulated solely at the mRNA level in the cell) mRNA measurement can be used to estimate cellular changes in response to external signals or environmental changes Measuring mRNA is therefore widely used to study gene expression The entire genome of an organism can be probed at a single point in time 3

4. Microarrays use base pairing - a process known as hybridization This helps identify unknown DNA sequences that might be present in a sample. C always pairs with G, and A always pairs with T or U www.affymetrix.com 4

5. Types of DNA Probes (reporter) on a microarray chip cDNA probesOligo probes cDNAs from high-throughput sequencing projects are PCR- verified then spotted directly from multiwell plates. Sequence ID is done for probes of interest after hybridization data are acquired. As in RNA blot experiments, DNA probes are not necessarily gene-specific. Oligo sequence design is done before the array is produced and experimentation is carried out  DNA probes are gene- specific by design. DNA probes are synthesized based on design specifications either directly on the fixed surface or prior to spotting onto the fixed surface. Long oligo probes Short oligo probes 50 -70-mers25-mers 5

6. ‘Spotted’ Array Technology – cDNA & Long Oligo DNA ‘probes’ are immobilized in grids or ‘arrays’ onto a small platform, commonly microscope slide. 6

7. The Microarray Technologies Spotted Microarray Affymetrix GeneChip cDNAs, clones, or short and long oligonucleotides deposited onto glass slides Each gene (or EST) represented by its purified PCR product Simultaneous analysis of two samples (treated vs untreated cells) provides internal control. short oligonucleotides synthesized in situ onto glass wafers Each gene represented multiple times - using 16-20 (preferably non-overlapping) 25-mers. Each oligonucleotide has single-base mismatch partner for internal control of hybridization specifity. relative gene expressions absolute gene expressions Each with its own advantages and disadvantages 7

8. Arrays for Molecular Ecology studies Anonymous array = probes used on the array are random (not based on known sequences). Useful for cross species studies Dedicated array = probes of genes that are specific to particular pathway or phenotype eg. Plant-herbivore interaction) 8

9. Spots and more spots….. http://www.liv.ac.uk/vets_med_images/lmf/microarray_spots_zoomed.jpg Software = Imagene for scoring the spots Looking for intensity differences Ratio based analysis 9

10. Data scoring Example of a heat map from microarray data Red = upregulated and Green = Down regulated genes Many ways to interpret the data but one should always back up suspect genes with other technique eg. Northerns or Real Time PCR 10

11. Concerns: Reproducibility of experiment = $$ hence not done much Data is difficult to exchange due to the lack of standardization in arrays Probe that are designed to detect the mRNA of a particular gene may be relying on genomic EST information that is incorrectly associated with that gene Helps if data is MIAME (Minimum Information About a Microarray Experiment) compliant The MicroArray and Gene Expression Data (MGED) group is working on the standardization of gene expression and relevant annotations Lots of statistical concerns and it is important to consult with a good statistician before embarking on microarray experiment…lots of $$$ involved 11

12. Transcriptome analysis 12

13. Example of environmental factor (CO 2 ambient vs elevated) on gene families http://www.sciencephoto.com/media/169131/enlarge 13

14. Comparative genomics of gene expression

15. Phylogeny of seven spruce species based upon AFLPs

16. Microarray experiment design 7 species, 3 individuals for each species 21 hybridizations total Species Individuals Cy3 Cy5

17. Spruce microarray 21,842 cDNAs 11,224 have BLAST hits of e -5 or less 2,402 have BLAST hits of e -50 or less  (Much work needs to be done on annotation)

18. Inference of lineage-specific expression differences -relative to mean over all species -”star” phylogeny assumed Diversifying or “Darwinian” selection No selection Stabilizing selection

19. The evolution of gene expression among seven spruce species: fractions of clones showing Darwinian selection vs. neutrality vs. stabilizing selection (n=21,334)

20. Evolution of gene expression in gene families involved with resistance (n=329) (n=88)(n=96) All clones (n=21,334)