Microarray Design Basics

MICROARRAY-TARGET SELECTION AND DESIGN Mr.Anand M. Bambhania,Roll No.3 Miss Pavithra Jeyakumar, Roll No.7 Date: 15th July, 2008

CONTENTS: 1. Introduction………………………………………………………………………….01 2. Stages involved……………………………………………………………………... 02 2.1 Experimental Design 2.2 Preparation of mRNA and cDNA 2.3 Hybridization 2.4 Image scanning 2.5 Data analysis 2.6 Biological confirmation 2.7 Deposition into databank 2.8 Analysis of data with related data 3. Using microarray for target identification and selection……………………………06 4. Advantages………………………………………………………………………….07 5. Disadvantages……………………………………………………………………….07 6. References…………………………………………………………………………..08 6.1 Bibliography 6.2 Webliography

1. Introduction:  A microarray is a solid support (ex. Glass slide or nylon membrane) on which DNA of known sequence is deposited in an regular grid like array.  The DNA may take the form of cDNA or oligonucleotides, although other materials(such as genomic DNA clones) may be deposited as well.  Several nanograms of DNA are immobilized on the surface of an array.  RNA is extracted from the biological sources of interest-also known as targets, such as cell lines with or without drug treatment, or tissues from wild type or mutant organisms.  The RNA(or mRNA) is often converted to cDNA, labeled with fluorescence or radioactivity and hybridized to the array. Page 1 of 8

 During this hybridization, cDNAs derived from RNA molecules in the biological starting material can hybridize selectively to their corresponding nucleic acids on the microarray surface.  Following washing of the microarray, image analysis and data analysis are performed to quantify the signals that are detected.

2. Stages involved:  An overview of the microarray procedures are given in figure below:

Stage 1:

Experimental design

Stage 2: RNA preparation and probe preparation Stage 3:

Comparison of two biological samples

Stage 4: Image analysis. Stage 5: Data analysis

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Stage 6: Biological confirmation

Stage 7: Data is deposited in an database

Stage 8: Further analysis

2.1

Experimental design: 

The experimental design of an microarray can be considered in three parts: 1) Biological sample comparison:  The biological sample are selected for comparison, such as cell lines with or without drug treatment.  If multiple samples are used, these are called “biological replictates”. 2) Extracting RNA, converting and labeling:  The RNA is extracted and labeled with radioactivity or fluorescence. 3) Arrangement of array elements on a surface:  The array elements are arranged in a randomized order.  In some cases, array elements are spotted in duplicate.

2.2

RNA preparation and probe preparation:  RNA can be purified from cells or tissues using reagents such as TRIzol.  In comparing two samples,it is essential to purify RNA under similar conditions.  The purity and quality of RNA should also be assessed spectrophotometrically by measuring a260/a280 ratio and by gel electrophoresis. Page 3 of 8

 Probe is generated and is labeled with fluorescence/ chemical dyes. 2.3

Hybridization:  In hybridization, two different samples are being used: (i) (ii)

Target sequence which consists of 100-2000 cDNA or oligonucleotides Probe sequence

 Target sequences are already fixed on glass slide or nylon membrane or silicon chips.  Probe sequences are complementary of gene which is to be analyzed and is labeled with radio activity or fluorescence.  Probes are added in target, kept overnight and washed away in morning. 2.4

Image analysis:  After washing, image analysis is performed to obtain a quantitative description of the extent to which each mRNA in the sample is expressed.  For experiments using radioactive probes, image analysis is performed by using quantitative phosphorimaging.  For fluorescence-based microarrays, the array is excited by a laser and fluorescence intensities are measured.  Data for Cy5 and Cy3 may be sequentially obtained and used to obtain gene expression ratios.

Figure 1: Example of microarray experiment using radioactive probes.

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Figure 2: Example of microarray expement using fluoroscently labeled sample. 2.5

Data analysis:  Analysis of microarray data is performed to identify individual genes that have been differentially regulated.  It is also used to identify broad patterns of gene expression.  Some statistical methods are also used for data analysis.

2.6

Biological confirmation:  Microarray experiments can be thought of as “hypothesis-generating” experiments.

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 The differential up-or down-regulation of specific genes can be measured using independent assays such as:  Northern blots  polymerase chain reaction (RT-PCR)  in situ hybridization 2.7

Deposition into Databases:  



2.8

Most academic researchers agree that microarray data should be deposited in public repositories upon publication for future reference. Such databases can be classified mainly into two types: (i) Microarray database – stores complete set of raw and processed data. (ii) Gene expression database – Mainly stores the expression of gene in tissues etc.

There are two main repositories: (i) Gene Expression Omnibus at NCBI. (ii) ArrayExpressat at the European Bioinformatics Institute (EBI).

Further analysis:  We can use stored data for further experiments.  It is likely that uniform standards will be adopted for all microarray experiments.  An ongoing trend in the field of bioinformatics is the unification and crossreferencing of many databases, such as has occurred for databases of molecular sequences and for databases of protein domains.

3. Using microarrays for target identification and selection:    

Many of the simple molecular pathways leading to disease have already been identified and addressed by research. Drugs have been created based on the identification of these pathways. But it is the ability to identify the complex pathways, such as those associated with cancer, which will allow new drugs to be developed. Drug developers can generate hypotheses for complex disease mechanisms with genome-wide expression profiling using DNA microarrays, allowing them to identify targets for drugs. Figure 1 shows the general idea of how DNA microarrays can be used to identify pathways. A diseased cell is profiled, and the microarray data is analyzed to discover pathways. Researchers can then target drugs to the affected areas in the biochemical pathways. Page 6 of 8

4. Major advantages:  Fast: One can obtain data on the expression levels of over 10,000 genes within a single week.  Comprehensive: The entire yeast genome can be represented on a chip.  Flexible: cDNA or oligonucleotides corresponding to any gene can be represented on a chip.

5. Major Disadvantages:  Cost: Many researchers find it prohibitively expensive to perform sufficient replicates and other controls.  Unknown significance of RNA: The final product of gene expression is protein, not RNA.  Uncertain quality control: It is impossible for most investigators to asses the identity of DNA immobilized on any microarray. Also, there are many artifacts associated with image analysis and data analysis.

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6. References: 6.1 Bibliography: 1) Bioinformatics and functional genomics, 2003 edition, Pevsner J. 6.2 Webliography: 1) http://pevsnerlab.kennedykrieger.org/ppts/2006-09-18_lect05_ch6.pdf 2) http://plasticdog.cheme.columbia.edu/undergraduate_research/projects/sahil_meht a_project/introduction.htm

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