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Micro-array study for gene expression

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History

cDNA microarrays have evolved from Southern blots, with cDNA microarrays have evolved from Southern blots, with clone libraries gridded out on nylon membrane filters clone libraries gridded out on nylon membrane filters being an important and still widely used intermediate. being an important and still widely used intermediate. Things took off with the introduction of non-porous solid Things took off with the introduction of non-porous solid supports, such as glass - these permitted miniaturization - supports, such as glass - these permitted miniaturization - and fluorescence based detection. Currently, about 20,000 and fluorescence based detection. Currently, about 20,000 cDNAs can be spotted onto a microscope slide. cDNAs can be spotted onto a microscope slide.

05/02/23 3Department of Statistics, University of California, Berkeley, and

Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

THE PROCESSTHE PROCESSBuilding the Chip:

MASSIVE PCR PCR PURIFICATION and PREPARATION

PREPARING SLIDES

PRINTING

Preparing RNA:

CELL CULTURE AND HARVEST

RNA ISOLATION

cDNA PRODUCTION

Hybing the Chip:POST PROCESSING

ARRAY HYBRIDIZATION

PROBE LABELING

DATA ANALYSIS

05/02/23 4Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

cDNA clones(probes)

PCR product amplificationpurification

printing

microarray Hybridise target to microarray

mRNA target)

excitation

laser 1laser 2

emission

scanning

analysis

overlay images and normalise

0.1nl/spot

05/02/23 5Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

Preparing RNA:

RNA ISOLATION

cDNA PRODUCTION

Designing experiments to profile conditions/perturbations/mutations and carefully controlled growth conditions

RNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip.

Single strand synthesis or amplification of RNA can be performed.cDNA production includes incorporation of Aminoallyl-dUTP.

Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

Preparing RNA:

RNA ISOLATION

cDNA PRODUCTION

Designing experiments to profile conditions/perturbations/mutations and carefully controlled growth conditions

RNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip.

Single strand synthesis or amplification of RNA can be performed.cDNA production includes incorporation of Aminoallyl-dUTP.

Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

Preparing RNA:

RNA ISOLATION

cDNA PRODUCTION

Designing experiments to obtain conditions necessary to induce changes in the plaque to cause its rupture

RNA yield and purity are determined by system.

Single strand synthesis or amplification of RNA can be performed.

OBTAINING THE TARGET TISSUE

05/02/23 6Department of Statistics, University of California, Berkeley, and

Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

Hybing the Chip:

ARRAY HYBRIDIZATION

PROBE LABELING

DATA ANALYSIS

Cy3 and Cy5 RNA samples are simultaneously hybridized to chip. Hybs are performed for 5-12 hours and then chips are washed.

Two RNA samples are labelled with Cy3 or Cy5 monofunctional dyes via a chemical coupling to AA-dUTP. Samples are purified using a PCR cleanup kit.

Ratio measurements are determined via quantification of 532 nm and 635 nm emission values. Data are uploaded to the appropriate database where statistical and other analyses can then be performed.

05/02/23 7Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

Biological Question

SamplepreparationMicroarray

Life Cycle

Data Analysis & Modeling

Microarray Reaction

MicroarrayDetection

Taken from Schena & Davis

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Schematic of probe preparation , hybridization, scanning and immage analysis

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Our Aim

•We intend to induce rupture of atherosclerotic plaque in apo-e mice by injecting several drugs, which we hope will cause rupture of the plaque by altering the hemodynamic system and raising the oxidative stress and alter the composition of the plaque.

•After achieving this goal we will perform gene-array study on these specimens and will try to find out which genes are expressed more in plaques which are ruptured or are prone to rupture, by comparing our results at the histopathology lab which will define the structural details of such vulnerable plaques.

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DNA Sequence Synthesis We are going to design a genearray slide based on We are going to design a genearray slide based on

oligonucleotide sequences that we will derive from oligonucleotide sequences that we will derive from the genes that we are interested in.the genes that we are interested in.

To do this, we will proceed as follows:To do this, we will proceed as follows: First we will gather the cDNA or mRNA sequences First we will gather the cDNA or mRNA sequences

of the desired genes from Entrez.of the desired genes from Entrez. Then we will pickup a sequence of genes from that Then we will pickup a sequence of genes from that

particular gene and by using Primer Premier particular gene and by using Primer Premier program we will design a primer to use as a program we will design a primer to use as a hybridization probe.hybridization probe.

05/02/23 12Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,

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We will order a probe to be made by a company that will We will order a probe to be made by a company that will make that on a commercial basis.make that on a commercial basis.

The micro array-chip, we will make at the lab by The micro array-chip, we will make at the lab by synthesizing cDNA from mRNA, that we isolated.synthesizing cDNA from mRNA, that we isolated.

We will obtain the tissue samples from the mice, according We will obtain the tissue samples from the mice, according to our study design, we will purify the mRNA according to our study design, we will purify the mRNA according the specific protocol. the specific protocol.

mRNA from the target will be converted into cDNA, and mRNA from the target will be converted into cDNA, and allowed to hybridiz on the microarray chip that we allowed to hybridiz on the microarray chip that we designed designed

The emission of colors will be captured by the scanner, The emission of colors will be captured by the scanner, and the results will be analysed depending upon their and the results will be analysed depending upon their intensity and presence and absence of colorintensity and presence and absence of color

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• Drugs being used to induce rupture ofatherosclerotic plaque in apo-e mice

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Drugs being used

• LNAME• Adrenalin• Cocaine• Xanthine• Xanthine Oxidase• Interleukin-1 beta• Methionine• Buthionine Sulfoximine

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If we do not find anything

Very pleasant, at least we shalll find something new