Microarray Technology

What Is A Microarray?

“A microarray is a tool for analyzing

gene expression that consists of a

small membrane or glass slide

containing samples of many genes

arranged in a regular pattern.”

Uses

• Analysis of gene expression

• SNP analysis

• MHC typing

• Identification of pathogens

• Identification of tumours

MICROARRAYS

• The underlying principle of microarray technology is the ability of DNA to bind to itself and to RNA.

• Analyzing gene expression involves the detection of mRNA species

(transcriptome) present in a cell or tissue at a particular point in time.

Microarrays

RNA RNA

Cy3 labelled

cDNA

Cy5 labelled

cDNA

Hybridization

Evolution of Microarrays

• One gene / one experiment– RT-PCR, northern blots, RNase assays

• Macroarrays– Nylon membranes

– 32P labelled samples

• Microarrays– Glass slides

– Fluorescent labelled samples

• Affymetrix

• There are 3 main types of microarrays

• Glass cDNA microarrays

• Glass oligonucleotide microarrays

• Affymetrix

Glass Microarrays

• cDNA arrays

– >1000 nucleotides in length

– cDNA libraries

• Oligonucleotide arrays

– 70-80 nucleotides in length

– Increased specificity

Affymetrix

• 25 nucleotides in length

• Mismatch pair

– Single mismatch at centre of probe

– Used to detect & eliminate false results

• 11 probe pairs per gene

• Photolithography

Bovine MicroarraysMicroarray Format Source of Material

BOTL cDNA Bovine total leukocyte cDNA library & immune

gene amplicons

Pyxis CattleArray 7600 cDNA Bovine spleen & placental cDNA libraries

NBFGC cDNA MARC & BARC cDNA libraries

BMAM cDNA MARC & BARC cDNA libraries

Mammary gland specific

Endometrial/placental

microarray

cDNA Bovine placental & endometrial cDNA libraries

Bovine immune-

endocrine microarray

cDNA Bovine immune & endocrine gene amplicons

Bovine MHC class I

microarray

oligos BoLA DRB3 & DQA sequences

BoMP microarray

RIGUA

cDNA

Oligos

Bovine macrophage cDNA library

Bovine immune & inflammatory gene amplicons

Affymetrix oligos Various cDNA libraries

ARK-Genomics cDNA Various cDNA libraries

DNA MICROARRAYS CAN BE USED TO DETECT DIFFERENCES

IN THE LEVELS GENE EXPRESSION IN DIFFERENT

POPULATIONS OF CELLS ON A GENOME-WIDE LEVEL.

GENE EXPRESSION = A GENE’S ABILITY TO MAKE A GENE

PRODUCT.

MOST GENES CODE FOR PROTEINS (PROTEIN ENCODING

GENES) THAT WHEN EXPRESSED, PRODUCE A GENE-SPECIFIC

PROTEIN.

DNA MICROARRAYS

• A small 1 square centimeter chip that’s divided into thousands of squares.

• Each square contains many copies of a single gene.

• Originally developed by Patrick Brown at the Stanford University School of Medicine to determine which genes are involved in yeast cell sporulation.

APPLICATIONS OF DNA MICROARRAY ANALYSIS.

• Genes that are involved in the development of an organism.

• Genes that are activated as a family of genes (gene expression

profiling).

• Genes that are involved in human cancers.

• Earlier diagnosis of human cancers.

EXAMPLE OF A CANCER SUBTYPE DETERMINED VIA DNA

MICROARRAY ANALYSIS.• Two subtypes of diffuse large B cell lymphoma.

• One group of tumor cells expressed a large group of genes that are involved in early B cell development.

• Another group of tumor cells expressed a large group of genes that are involved in late B cell development.

Microarrays have many uses.

1. Major use is to investigate gene expression.

2. mRNA isolated from a cell gives a snap-shot of

the events occurring at that time.