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A Paper on Dna Microchip-By
Artigo Modulo 1 Brief Bioinform
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MICROARRAY-TARGET SELECTION AND DESIGN Mr.Anand M. Bambhania,Roll No.3 Miss Pavithra Jeyakumar, Roll No.7 th
Date: 15 July, 2008
CONTENTS:
1. Introduction………………………………………………………………………… Introduction………………………………………………………………………… 2. Stages involved…………………………………………………………………… involved…………………………………………………………………… 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…………………………… selection…………………………… 4. Advantages………………………………………………………………………… Advantages ………………………………………………………………………… 5. Disadvantages……………………………………………………………………… Disadvantages ……………………………………………………………………… 6. References………………………………………………………………………… References ………………………………………………………………………… 6.1 Bibliography 6.2 Webliography Sign up to vote on this title
1. Introduction:
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During this hybridization, cDNAs derived from RNA molecules in biological starting material can hybridize selectively to their correspon nucleic acids on the microarray surface. Following washing of the microarray, image analysis and data analysis performed to quantify the signals that are detected.
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 Sign up to vote on this title
Stage 4: Image analysis.
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A Paper on Dna Microchip-By
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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 part
1) Biological sample comparison: The biological sample are selected for comparison, such as lines with or without drug treatment. If multiple samples are used, these are called “biolo replictates” replictates”. 2) Extracting RNA, converting and labeling: Sign up to vote on this title The RNA is extracted and labeled with radioactivity Useful Not useful fluorescence. 3) Arrangement of array elements on a surface:
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Probe is generated and is labeled with fluorescence/ chemical dyes.
Hybridization:
In hybridization, two different samples are being used: (i) (ii)
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Target sequence which consists of 100-2000 cDNA or oligonucleotides Probe sequence
Target sequences are already fixed on glass slide or nylon membrane or sil chips. Probe sequences are complementary of gene which is to be analyzed an labeled with radio activity or fluorescence. Probes are added in target, kept overnight and washed away in morning.
Image analysis:
After washing, image analysis is performed to obtain a quantitative descrip of the extent to which each mRNA in the sample is expressed. For experiments using radioactive probes, image analysis is performe using quantitative phosphorimaging. phosphorimaging. For fluorescence-based microarrays, the array is excited by a laser fluorescence intensities are measured. Data for Cy5 and Cy3 may be sequentially obtained and used to obtain g expression ratios.
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Figure 2: Example of microarray expement using fluoroscently labe sample. 2.5
Data analysis:
Analysis of microarray data is performed to identify individual genes that been differentially regulated. It is also used to identify broad patterns of gene expression. expression. Some statistical methods are also used for data analysis.
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Northern blots polymerase chain reaction (RT-PCR) in situ hybridization
Deposition into Databases:
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The differential up-or down-regulation of specific genes can be meas using independent assays such as:
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Most academic researchers agree that microarray data should be deposite public repositories upon publication for future reference. Such databases can be classified mainly into two types: – stores complete set of raw and proce (i) Microarray database – stores data. (ii) Gene expression database – Mainly stores the expressio gene in tissues etc.
There are two main repositories: (i) Gene Expression Omnibus at NCBI. (ii) ArrayExpressat ArrayExpressat at the t he European Bioinformatics Institute (E
Further analysis: We can use stored data for f or further experiments. It is likely that uniform standards will be adopted for all microa experiments. An ongoing trend in the field of bioinformatics is the unification and cr referencing of many databases, such as has occurred for databases molecular sequences and for databases of protein domains. Sign up to vote on this title
Not useful Usefuland selection: 3. Using microarrays for target identification
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4. Major advantages:
Fast: One can obtain data on the expression levels of over 10,000 genes with single week. Comprehensive: The entire yeast genome can be represented on a chip. Sign up to this title Flexible: cDNA or oligonucleotides corresponding tovote anyongene can be represe on a chip. Useful Not useful
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6. References: 6.1 Bibliography:
1) Bioinformatics and functional genomics, 2003 edition, Pevsner J. 6.2 Webliography:
