Microarray hybridization

• Usually comparative– Ratio between two

samples

• Examples– Tumor vs. normal tissue– Drug treatment vs. no

treatment– Embryo vs. adult

mRNA

cDNA

DNAmicroarray

samples

2. Spotting DNA on the chip

Capillary action on contact

Apply small droplets on contact

Pin stamps DNA on contact

Spray droplets under pressure

Types of printing pins

Synthesis of High Density Oligonucleotide Arrays by Photolithography/Photochemistry

Comparisons of microarrays

Scanning of microarrays

• Confocal laser scanning microscopy

• Laser beam excites each spot of DNA

• Amount of fluorescence detected

• Different lasers used for different wavelengths– Cy3– Cy5

laserdetection

Spotting of PCR amplified clones using MicroGrid II 610

Hybridization of target with spotted slides by GeneTAC Hybstation Quantification of Cy3 & Cy5 labeled target using Nanodrop

Control and water Stressed Cotton plants

RNA Isolation

cDNA Library construction

PCR amplification of clones

Spotting of slides with PCR amplified clones using MicroGrid II 610

Preparation of Cy3 and Cy5 labeled TARGET

Hybridization of target with spotted slides by GeneTAC Hybstation

Scanning of slides by GeneTAC UC4X4

Slide Image after hybridization

4.1 Scanning & capturing images

Image files of hybridized slide in both Cy3 and Cy5 channels

Up-regulated

Down-regulated

Co-expressed

ScanningHybridized Microarray

Laser 2Laser 1

Monochrome pictures combined

Emission

Excitation

(two-color arrays)

Image Segmentation

Scanned Image

Numerical Data

Segmentation Software

• Results given as ratios• Images use colors:

Cy3 = GreenCy5 = redYellow

– Yellow is equal intensity or no change in expression

Analysis of hybridization

Intensity-dependent bias

A

M = log(Cy3/Cy5)

Low intensities

M<0: Cy3<Cy5

High intensities

M>0: Cy3>Cy5

* Global normalization cannot remove intensity-dependent biases

A

We expect the M vs A plot to look like:

M = log(Cy3/Cy5

)

LOWESS (Locally Weighted Scatterplot Smoothing)

• Local linear regression model

• Tri-cube weight function

• Least Squares

Estimated values of log2(Cy5/Cy3) as function of log10(Cy3*Cy5)

Heat map of Affymetrix 430 V2.0 GeneChip dataHeat map of Affymetrix 430 V2.0 GeneChip dataPotential transducers of TPA effects

Gene ID CON TPA T+A Gene Identifier

MAPK signaling pathwayFgf23 14.6 421.9 120.3 AF263536Pla2g2d 353.1 1159.6 384.9 AF169407Dusp9 140.4 333.1 146.8 AV295798Mknk2 1387.4 2855.1 1815.1 NM_021462Prkx 1992.4 3507.8 2156.1 BB367549Fgf5 2626.4 5019.5 2426.4 AV240088Tgfbr1 216.8 355.8 195.6 NM_009370Braf 2965.3 4573.2 2721.6 BM217816Prkce 206.7 330.5 173.6 BB534545Ppp3r1 185.6 294.3 141 NM_024459Map2k7 214.2 326.5 161.8 NM_011944Nfatc4 1831.7 1144.5 2288.8 BF227641Rasgrp1 922 577.8 1131.1 BE691356Rps6ka5 829.5 535.3 872.8 BQ174267Tgfb2 1562.7 913.4 1886 BF144658Fgf13 3698 2148.8 4445.1 BC018238Pdgfra 1359.9 665.1 1916.1 M57683Il1r2 8389.2 4262.1 10932 NM_010555Fgf7 1964.5 1022.6 2003.4 NM_008008Fgf11 336 160.8 375.2 AW488620Acvr1c 484.9 222.7 561.1 BB396526Tgfb3 1324.7 580.2 1334 BC014690Mef2c 969.4 622.7 951.6 BC026841Map2k2 774.3 479 719.1 AW542161Egf 847 481.7 825.5 NM_010113Rac2 4733.7 2495.4 4118.5 NM_009008Nfatc2 2110.1 1132.6 1781.9 BM122872Ptpn5 686.5 316.8 550.3 BB188812Ptprr 339 115.8 279.1 AF129509Hspb2 1932.8 741.9 1167.4 AK012780Nf1 377.5 127 192.5 BB202321Bdnf 224.6 34.2 182.4 NM_007540

Cytokine-cytokine receptor interactionTnfrsf19 309.8 880.8 347.1 AF167552Tnfrsf9 165.7 409.2 96.5 NM_011612Tgfbr1 216.8 355.8 195.6 NM_009370Kitl 538.8 890.6 479.1 BC011322Prlr 704.3 1061.1 671.2 BC005555Il2ra 79.9 26.3 427.8 M30856Ccl11 2425.3 1568.1 3923.1 NM_011330Xcl1 520.9 318.2 853.3 NM_008510Ccl6 26401 16280.6 42110.5 BC002073Lifr 655.3 395.8 934.3 D17444Tnfsf13 2934.4 1937.3 3583.2 NM_023517Cxcl4 22326 14790.7 25337.9 NM_019932Ifnar1 447.5 291.1 482.2 BB206335Tgfb2 1562.7 913.4 1886 BF144658Il17r 1843.8 1179.1 1895.8 NM_008359Tnfrsf5 549.1 299.2 704.3 AI385482