Microarray hybridization
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samples. mRNA. cDNA. DNA microarray. Microarray hybridization. Usually comparative Ratio between two samples Examples Tumor vs. normal tissue Drug treatment vs. no treatment Embryo vs. adult. 2. Spotting DNA on the chip. Types of printing pins. Apply small droplets on contact.

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Microarray hybridization

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Microarray hybridization

samples

mRNA

cDNA

DNA

microarray

Microarray hybridization

  • Usually comparative

    • Ratio between two samples

  • Examples

    • Tumor vs. normal tissue

    • Drug treatment vs. no treatment

    • Embryo vs. adult


2 spotting dna on the chip

2. Spotting DNA on the chip


Microarray hybridization

Types of printing pins

Apply small droplets on contact

Capillary action on contact

Spray droplets under pressure

Pin stamps DNA on contact


Microarray hybridization

Synthesis of High Density Oligonucleotide Arrays by Photolithography/Photochemistry


Comparisons of microarrays

Comparisons of microarrays


Scanning of microarrays

Scanning of microarrays

laser

  • Confocal laser scanning microscopy

  • Laser beam excites each spot of DNA

  • Amount of fluorescence detected

  • Different lasers used for different wavelengths

    • Cy3

    • Cy5

detection


Microarray hybridization

Spotting of PCR amplified clones using MicroGrid II 610


Target preparation and hybridization

Target Preparation and Hybridization

Hybridization of target with spotted slides by GeneTAC Hybstation

Quantification of Cy3 & Cy5 labeled target using Nanodrop


Microarray hybridization

DIFFERENT STEPS IN HIGH THROUGHPUT STUDIES OF PLANT GENES

Control and water Stressed Cotton plants

cDNA Library construction

RNA Isolation

PCR amplification of clones

Preparation of Cy3 and Cy5 labeled TARGET

Spotting of slides with PCR amplified clones using MicroGrid II 610

Hybridization of target with spotted slides by GeneTAC Hybstation

Slide Image after hybridization

Scanning of slides by GeneTAC UC4X4


4 1 scanning capturing images

4.1 Scanning & capturing images


Scanning of hybridized slides

Scanning of Hybridized Slides

Co-expressed

Up-regulated

Down-regulated

Image files of hybridized slide in both Cy3 and Cy5 channels


Scanning

Scanning

Hybridized Microarray

Excitation

Laser 1

Laser 2

Emission

Monochrome pictures combined

(two-color arrays)


Image segmentation

Image Segmentation

Numerical Data

Scanned Image

Segmentation Software


Analysis of hybridization

Analysis of hybridization

  • Results given as ratios

  • Images use colors:

    Cy3 = Green

    Cy5 = red

    Yellow

    • Yellow is equal intensity or no change in expression


Intensity dependent bias

Intensity-dependent bias

M>0: Cy3>Cy5

M<0: Cy3<Cy5

Low intensities

High intensities

M = log(Cy3/Cy5)

* Global normalization cannot remove intensity-dependent biases

A


Microarray hybridization

We expect the M vs A plot to look like:

M = log(Cy3/Cy5)

A


Lowess locally weighted scatterplot smoothing

LOWESS (Locally Weighted Scatterplot Smoothing)

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

  • Local linear regression model

  • Tri-cube weight function

  • Least Squares


Microarray hybridization

Heat map of Affymetrix 430 V2.0 GeneChip data


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