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

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

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•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

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.com4

Types of DNA Probes (reporter) on a microarray chip

cDNA probes Oligo 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 probes50 -70-mers 25-mers

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‘‘Spotted’ Array Technology – cDNA & Long Spotted’ Array Technology – cDNA & Long OligoOligo

DNA ‘probes’ are immobilized in grids or ‘arrays’ onto a small platform, commonly microscope slide.

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The Microarray TechnologiesThe Microarray Technologies

Spotted MicroarraySpotted Microarray Affymetrix GeneChipAffymetrix 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 situonto 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

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)

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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

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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

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

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Transcriptome analysis

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Example of environmental

factor (CO2 ambient vs

elevated) on gene families

http://www.sciencephoto.com/media/169131/enlarge13

Comparative genomics of Comparative genomics of gene expressiongene expression

Phylogeny of seven spruce species based upon AFLPs

Microarray experiment design 7 species, 3 individuals for each species 21 hybridizations total

Species

Individuals

Cy3 Cy5

Spruce microarraySpruce 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)

Inference of lineage-specific expression differences-relative to mean over all species-”star” phylogeny assumed

Clone

Lineage a

Lineage b

Lineage c

Lineage d

Lineage e

Lineage f

Lineage g

Species variance

Error variance

Species /total

chi-square (6 df)

WS0262_L03 -0.03 -0.01 0.18 -0.24 -0.12 0.19 0.03 0.024 0.001 0.972 206.558 WS0047_G13 -0.10 0.07 0.09 -0.17 0.27 0.17 -0.32 0.043 0.001 0.972 206.558 WS01027_A02 0.05 0.05 -0.22 -0.04 0.15 0.15 -0.13 0.020 0.001 0.971 203.785 WS00723_G05 0.05 -0.44 0.02 -0.02 0.06 -0.17 0.50 0.080 0.002 0.971 203.785 … … WS0061_B08 0.02 -0.08 0.08 0.07 0.22 -0.17 -0.13 0.019 0.019 0.497 5.917 WS0053_E12 0.13 -0.20 0.21 0.05 -0.32 -0.12 0.26 0.048 0.049 0.496 5.917 WS0085_A17 -0.20 -0.05 -0.17 0.02 0.12 0.19 0.08 0.021 0.022 0.496 5.916 WS0038_D14 -0.22 0.04 0.17 0.05 0.46 -0.20 -0.30 0.071 0.072 0.496 5.913 WS0093_M07 0.05 0.02 0.06 -0.20 -0.08 0.09 0.05 0.011 0.011 0.496 5.909 … … WS00937_G12 0.09 0.01 -0.01 0.06 -0.25 0.23 -0.12 0.023 0.721 0.031 0.195 WS01010_O12 -0.52 0.04 -0.03 -0.14 0.55 -0.03 0.13 0.101 3.128 0.031 0.194 WS00722_B02 -0.08 -0.19 0.30 -0.23 -0.20 -0.30 0.69 0.132 4.129 0.031 0.191 WS01028_C20 -0.15 0.15 -0.01 -0.07 0.00 0.16 -0.07 0.013 0.425 0.031 0.190 WS01014_I04 -0.01 -0.07 0.07 0.07 -0.03 -0.01 -0.01 0.003 0.088 0.031 0.189

Diversifying or “Darwinian” selection

No selection

Stabilizing selection

The evolution of gene expression among seven spruce species: fractions of clones showing Darwinian selection vs. neutrality vs. stabilizing selection

DarwinianNeutralStabilizing

DarwinianNeutralStabilizing

All genes

42%

50%

8%

(n=21,334)

Evolution of gene expression in gene families involved with resistance

P450 genes

42%

48%

10%

Terpenoid genes

54%39%

7%

Phenylpropanoid genes

44%

47%

9%

All genes

42%

50%

8%

DarwinianNeutralStabilizing

DarwinianNeutralStabilizing

(n=329)

(n=88)(n=96)

All clones (n=21,334)