Overview of Microarray

2/71Gene Expression

Gene expression

Production of mRNA is very much a reflection of the activity level of gene

In the past, looking at whether a specific gene is turned up (upregulated) or turned off (downregulated) under certain condition

3/71Microarray Data

Microarray: a technological advancement study the genes of an organism’s at once

Microarrays are a massively-parallel Northern Blot

High throughput method allow for the global study of changes in gene expression

→ complete cellular snapshot

4/71Reverse Transcription

Clone cDNA strands, complementary to the mRNA

5/71Microarray Experiments

mRNA levels compared in many different contexts

Different tissues, same organism (brain vs. liver)

Same tissue, same organism (tumor vs. non-tumor)

Same tissue, different organisms (wild-type or mutant)

Time course experiments (development)

6/71Expression Profiles in Tissues

In any type of tissue, only a limited set of the genes are switched on at any given time.

Also, from one tissue type to another, the limited set of genes involved will vary.

Thus, each tissue can be identified by its unique pattern of gene expression. This pattern is often called an “expression profile” or a “molecular signature”.

Here is an example of a normal breast cell and a normal prostate cell. Although both of these cells have many mRNAs and proteins in common (grey), they also have unique differences.

7/71Expression Profiles in Cancer

It is possible to measure differences between a normal and a cancer tissue of the same type--- for example, normal and cancerous prostate.

When a normal prostate tissue is transformed into cancerous prostate tissue, the expression profile changes.

Any changes in gene expression ultimately cause alterations in protein production.

New expression profiles in a cancer cell can dramatically alter the network of proteins that interact.

A critical protein may no longer be available, another may be overproduced, yet another may be flawed. And when new genes become activated, entirely new proteins may be introduced.

Many different combinations of gene changes and protein interactions are seen in cancerous tissue.

8/71Cells and Gene Expression

The repertoire of gene products produced by a cancer cell might differ in two ways from its normal counterpart:Quantitatively As shown for gene B , which is expressed at an abnormally high level, and gene A, which is not expressed at all.

Qualitatively As shown for gene C*, which is mutated such that it produces an altered gene product.

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Two Main Technologies for Making Microarrays

Robotic spotting

From D. Steke’s Microarray Bioinformatics

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nylon array10pmol/mm2

glass array0.1pmol/mm2

From prof. 陳同孝’ s slide

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Two Main Technologies for Making Microarrays (cont’d)

In situ synthesis Using photolithography

http://www.affymetrix.com/technology/manufacturing/index.affx

12/71Two Type of Microarrays

(Harrington et al. 2000)

cDNA Array Oligo. Array

13/71cDNA Array

(Harrington et al. 2000)

14/71cDNA Probe Preparation

http://www.accessexcellence.org/AB/GG/polymerase.html

15/71Sample Preparation

Compare the genetic expression in two samples of cells

SAMPLES

cDNA labelled red/green

16/71Dye Labelling

aminoally-dUTP

17/71Hybridization and Scanning

HYBRIDIZE

Add equal amounts of labelled cDNA samples to microarray.

SCAN

Expression profiling using cDNA microarraysNature, 21, 1999

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http://www.iridian.ca/products/products_fluorescence.htm

19/71cDNA Array (cont’d)

cDNA Array: e.g. Agilent One probe one gene Processing steps:

Experimental design Sample preparation RNA extraction Prepare cDNA Labeling cDNA with dye Hybridization Quantitation

Hybridization

Microarraymanufacturing

Samplepreparation

Quantitation

Data analysis

ExperimentProbe preparation

20/71cDNA Array (cont’d)

http://www.bioteach.ubc.ca/MolecularBiology/microarray/

21/71Oligonucleotide Array Oligonucleotide Array

Synthesized on a chip: e.g. Affymetrix Using photolithography Each gene may have several probe sets;

each probe sets have above 10 probes.

http://www.affymetrix.com/technology/manufacturing/index.affx

22/71Cross-Hybridization

23/71PM/MM

24/71PM/MM (cont’d)

25/71Oligonucleotide Array (cont’d)

http://www.bioteach.ubc.ca/MolecularBiology/microarray/

26/71Oligonucleotide Array (cont’d)

27/71Oligonucleotide Array (cont’d)

http://www.bioteach.ubc.ca/MolecularBiology/microarray/

28/71cDNA Array vs. Oligo. Array

Probes are cDNA fragments, usually amplified by PCR.

At least two samples are hybridized to chip.

One probe one gene.

Probes of varying length

Fluorescence at different wavelengths measured by a scanner.

Probes are deposited on a solid support, either positively charged nylon or glass slide.

Probes are oligos synthesized in situ using a photolithographic approach.

One target sample per array.

16-20 probe-pairs per gene.

Probes are 25-mers.

The apparatus requires a fluidics station for hybridization and a special scanner.

There are at least 5 oligos per cDNA, plus an equal number of negative controls.

From Dr. 吳漢銘’ s slide

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Advantages and Disadvantages of cDNA Array compared with Oligo. Array

Advantages Can choose the DNA on the array

Cheaper

Fluorescence at different wavelengths measured by a scanner

Can hybridize closely related species

Disadvantages Less specificity (will cross hybridize to genes ~80% homology) Cannot distinguish closely related gene families May need to confirm DNA sequence Repeated amplification and quality control

From Dr. 吳漢銘’ s slide

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Advantages and Disadvantages of cDNA Array compared with Oligo. ArrayAdvantages High specificity (small probe length means gene family members can be

differentiated) Very robust protocols and results are very reproducible Can use small amount of RNA Widely used, so annotation of probe sets is of relatively high quality

Disadvantages Very expensive to design (~US$300,000) Expensive to perform experiments (~US$400 + $300 labeling/hybridization) Limited to the species for which there are chips available sequence required Single target hybridization, so comparison always involves two experiments,

and dye swaps are impossible Match/mismatch technology has major limitations: mismatch signal often

higher than match, and dose response curve is different for each pair

From Dr. 吳漢銘’ s slide

31/71Microarray Experimental Flowchart

R=Rf-RbG=Gf-Gb

M=log2R/GA=1/2 log2RG

Microarray

Life Cycle