MICROARRAY TECHNOLOGY

BACKGROUND: NEED FOR MICROARRAY TECHNOLOGY:
A cell functions by using its genes to produce proteins and although each cell within an organism will usually contain the same set of genes, there are significant differences in which genes are activated and how they are controlled because a fraction of these genes are turned on and it is the subset that is "expressed" that confers unique properties to each cell type. "Gene expression" is the term used to describe the transcription of the information contained within the DNA. Scientists study the kinds and amounts of mRNA produced by a cell to learn which genes are expressed, which in turn provides insights into how the cell responds to its changing needs. Gene expression acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. For many years, study of a gene in this manner had to be done individually—looking at whether a specific gene is turned on (upregulated) or turned off (downregulated) under certain conditions. MICROARRAY is a technological advancement that has allowed scientists to study many, if not all, the genes of an organism’s at once. This high throughput method allows for the global study of changes in gene expression, giving us a complete cellular snapshot.
INTRODUCTION
Microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. The supports themselves are usually glass microscope slides, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support. It is a high throughput technology that allows simultaneous detection of thousands of genes.
In formal terms a microarray can be defined as:
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.
The American Heritage Dictionary defines "array" as "to place in an orderly arrangement". It is important that the gene sequences in a microarray are attached to their support in a fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides. Microarrays are also referred to as DNA arrays, DNA chips, biochips and GeneChips.
Microarrays come up with the advantageous features as:
– Multiplexing: handling multiple samples at a time
– Parallelism: Studying Thousands of genes simultaneously
– Miniaturization: small sized chip




PRINCIPLE:

The principle of microarrays is based upon base paired hybridization probing, a technique that uses fluorescently labeled nucleic acid molecules as "mobile probes" to identify complementary molecules, and sequences that are able to base-pair with one another.
PARAMETERS DETERMINING THE NATURE OF A MICROARRAY TECHNOLOGY

PARAMETER OPTION
Probes: features arrayed on the microarray substrate that have known identity or sequence cDNA, oligonucleotides, proteins, peptide nucleic acids, small molecules, cells, tissues, and organisms
Fabrication: techniques to array probes on the microarray substrate In situ synthesis, robotic deposition
Targets: samples to be analyzed against the probes DNA, mRNA, proteins, enzymes, small molecules
Assays: principles based on which the targets are being analyzed Hybridization, electrophoresis, flow cytometry, ELISA
Signal readout: principles based on which the assay results can be detected Fluorescence , chemiluminescence, mass spectrometry, radioactivity, electrochemistry
Image processing: signal intensities of hybridized array spots are quantified from the scanning image Software for image processing
Informatics: computational tools with which the huge amount of data generated from a microarray experiment can be effectively stored and interpreted Database management system, data mining and visualization, interpretation of biological meaning

There are various types of microarrays, and to name a few, they include: DNA Microarrays, Protein Microarrays, Tissue Microarrays, Cellular Microarrays and Antibody Microarrays. Among the various types, DNA microarrays, protein microarrays and tissue microarrays are most widely used. The focus in the following sections will be on DNA microarrays.

















DNA MICROARRAYS














DNA Microarray is the most widely used microarray technology. It is a type of nucleic acid-based multiplex technique involving high-density arrays of nucleic acids on glass that allows evaluating mRNA abundance of up to tens of thousands of genes simultaneously.
A DNA microarray consists of an orderly arrangement of DNA fragments representing the genes of an organism. Each DNA fragment representing a gene is assigned a specific location on the array, usually a glass slide, and then microscopically spotted (less than 1 mm) to that location. Through the use of highly accurate robotic spotters, over 30,000 spots can be placed on one slide, allowing molecular biologists to analyze virtually every gene present in a genome. The main advantage of microarrays is that the spots are single stranded DNA fragments that are strongly attached to the slide, allowing cellular DNA or RNA to be fluorescently labeled and laid overtop of the array. DNA or RNA in the overlaid sample will stick (through hybridization) to a complementary spot on the array, that is, Gene-A will stick to a spot composed of a Gene-A fragment. By exposing the microarray to a fluorescently labeled sample the DNA that hybridizes will be identifiable as glowing spots on the array, while the spots that have nothing hybridized to them will not be visible.
TYPES OF DNA MICROARRAYS
Two main types of commercial DNA microarrays are: oligonucleotide arrays and cDNA arrays.
1. OLIGONUCLEOTIDE ARRAYS
Oligonucleotide arrays (trademarked as a GeneChip by Affymetrix) use small 25 base pair gene fragments as the DNA probes to be spotted onto an array. These oligonucleotide probes are synthesized either in situ (on-chip) using photolithography or by conventional synthesis followed by on-chip immobilization.
APPLICATIONS OF OLIGONUCLEOTIDE ARRAYS
Oligonucelotide arrays are used for quantification of the amount of mRNA in a single sample (e.g. to determine the amount of mutated vs. non-mutated mRNA) or the comparison of two different samples hybridized to two separate arrays.
OLIGONUCLEOTIDE ARRAY TARGETS
Most array experimenters do not have access to micrograms of total RNA from their biological sample. A linear amplification strategy is highly recommended to obtain sample to be used for hybridization (targets). This technique consists of the following steps:


• First strand cDNA synthesis using reverse transcriptases;
• Generation of second strand cDNA using DNA polymerases; and
• In vitro transcription of the second strand cDNA to generate antisense RNA (aRNA) or complementary RNA (cRNA).
The probes are selected to have little cross-reactivity with other genes (targets) so that non-specific hybridization will be minimized.
2. cDNA ARRAYS
A cDNA array (also known as DNA microarray) is a different technology using the same principle; the probes in this case are larger pieces of DNA that are complementary to the genes one is interested in studying. With the cDNA probes prepared they can be mechanically spotted (robot spotting) onto a glass slide, normally in duplicate to serve as controls.
cDNA PROBES
• cDNA probes for making the array can also be generated from a commercially available cDNA library ensuring a close representation of the entire genome of an organism on the array.
• In an alternate target synthesis protocol, the target is generated by reverse transcription of the total RNA or the aRNA.
• Alternatively, PCR using specific primers can be used to amplify specific genes from genomic DNA to generate the cDNA probes.
When using cDNA spotted microarrays, two pools of RNA can be compared on the same array simultaneously. This is unlike Affymetrix GeneChip arrays, where RNA isolated from a single source is hybridized to an array or multiple arrays are required for comparison between more than one RNA samples.








DESIGN AND WORKING OF MICROARRAYS












DESIGN OF MICROARRAYS
Micro-array probes are synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization.
• Micro-arrays are printed in situ (on chip) using a combination of photolithography and combinatorial chemistry.
• For on-chip immobilization, probes can be mechanically spotted onto a glass slide by using robot.
PHOTOLITHOGRAPHY:
The production begins with a 5-inch square of quartz wafer.
This wafer is washed with a blocking compound that can be removed by exposure to light. What is needed next is referred to as a ‘mask’, which is designed with 18-20 micron square windows that allow light to pass through areas where a specific nucleotide is needed.
EXAMPLE:
If the mask is designed for the addition of thymine to the probe, you will find a window at that location, while probe locations where another base is required are blocked. Such a mask is laid on top of the array and ultraviolet light is shone onto it.
Probe locations that have windows are exposed to the light and the blocking compound removed from that probe, while covered locations still have the blocking compound present.
The quartz wafer is then washed with a solution of the desired nucleotide (e.g. thymine) that is linked to the same blocking compound, causing the nucleotide to attach to the probes that were exposed to light, while the nucleotide-attached blocking compound ensures that all of the probes are protected again. A capping step is added so that probes that did not attach their appropriate nucleotide are not incorrectly synthesized. The array is now ready for a new mask and the addition of a new nucleotide (cytosine in the figure). The process is repeated until all of the probes are complete (25 nucleotides each). Computer algorithms can calculate the optimal design of the masks that will minimize the number of reactions needed.
Once constructed, a microarray is ready for use in the laboratory.
WORKING OF MICROARRAYS
1. ISOLATION OF RNA FROM BIOLOGICAL SAMPLES

For microarray analysis, RNA can be isolated from a diverse set of biological samples using a combined phenol and glass-filter purification protocol. Each sample will contain thousands of different mRNA sequences representing all of the genes expressed in those cells.

2. GENERATION OF LABELED TARGETS:

Microarrays require 10-30 micrograms of labeled target per hybridization. Either labeled cDNA or aRNA (antisense) can be used for microarray experiments. In the Affymetrix system, the dNTPs are biotinylated, and later detection is performed with fluorescent streptavidin staining. In the simplest case, reverse transcription is performed using fluorescently tagged (e.g., Cy3 or Cy5), dinucleotide triphosphates (dNTPs), resulting in the generation of fluorescent cDNA (cDNA arrays).

2. TARGET HYBRIDIZATION.
The fluorescently labeled “targets,” are hybridized to gene-specific “probes.” Each target anneals to its corresponding probe spot on the microarray. The probes can be spotted cDNAs or oligonucleotides, or oligonucleotides that were synthesized directly on the micro-array surface. Stringent conditions are used to ensure that the probe sequences are entirely complementary to the micro-array spot sequences.
3. WASHING OF UNBOUND TARGETS
The slide is washed off to remove excess fluorescent targets not bound to spots.
4. DETECTION OF SIGNAL AND ANALYSIS OF DATA
After hybridization, a laser scanner is used to detect the specific fluorescence at each spot at two different wavelengths for green Cy3 or red Cy5. The colors of the spots are as follows:
Green = bound to Cy3-labeled cDNA, Red = bound to Cy5-labeled cDNA and Yellow = bound to both Cy3 and Cy5-labeled cDNA (these represent genes such as “housekeeping genes” that are required by all cells)
Software associated with the scanner allows identification of individual spots and measures each spot intensity. If all goes well, fluorescence intensity is proportional to the concentration of the relevant mRNA in the original sample.

DIAGRAMATIC REPRESENTATION OF MICROARRAY EXPERIMENT

The microarray is scanned with a laser beam, first at one wavelength to collect fluorescence data representing one probe, and then is scanned at a second wavelength to collect data representing the second probe. A computer compares the amount of fluorescence at each spot on the microarray for each probe. Through the use of computer software, the ratio of fluorescence is obtained and correlated with the clone address so the investigator knows which gene (spot on the slide) was expressed more in treated tissue as compared to control tissue.

















OTHER TYPES OF MICROARRAYS













Apart from DNA microarrays, protein microarrays and tissue microarrays are the types most widely used.
1. TISSUE MICROARRAYS
Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. Tissue microarrays (TMA) consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.
Tissue microarrays are different from DNA microarrays where each spot on an array represents a cloned cDNA or oligonucleotide that binds to the target sequence. With tissue microarrays, each array has patient specific histological samples from cancer infected tissues. The tissue microarray technique is best suited for screening one genetic marker or protein across thousands of samples where as DNA microarrays are best suited to study gene expression across thousands of genes.
2. PROTEIN MICROARRAYS
In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyze antibody–antigen, protein–protein, protein–nucleic-acid, protein–lipid and protein–small-molecule interactions, as well as enzyme–substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyze entire proteomes.
















APPLICATIONS OF MICROARRAYS














Microarrays can be used for large number of applications where high-throughput is needed. The current scope of microarray applications includes sequencing by hybridization, resequencing, mutation detection, assessment of gene copy number, comparative genome hybridization, and drug discovery and expression analysis.
1. SEQUENCE ANALYSIS
One of the earliest applications for microarrays was sequencing by hybridization. It is possible to determine the sequence of a target from the target’s pattern of hybridization with an array of all possible combinations of an oligonucleotide of a particular length. Subsequently, many other sequence-based applications have been developed, including genotyping, detection of point mutations, single nucleotide polymorphisms, and insertions and deletions, as well as the detection, identification, and enumeration of microorganisms.
2. GENE EXPRESSION ANALYSIS: GENE DISCOVERY
One of the most important applications of DNA Microarrays is the monitoring of gene expression where the abundance of the mRNA produced is determined for each gene. Such differences in gene expression are accountable for morphological and phenotypic differences as well as indicative of cellular responses to environmental stimuli and perturbations. Changes in the multi-gene patterns of expression can provide clues about regulatory mechanisms and broader cellular functions and biochemical pathways. In the context of medicine and treatment, such changes in patterns of expression can help to determine the causes and consequences of disease, how drugs and drug candidates work in cells and organisms.
Microarray technology has revolutionized studies of gene expression. A cDNA or oligonucleotide array complementary to expressed mRNA provides a means to simultaneously assess the expression of thousands of genes. Two-color labeling strategies introduce an additional degree of sophistication because they facilitate simultaneous comparison between gene expression in control and test cells on the same array. A goal in expression analysis is to place all of the genes for an organism on a single chip and then use that microarray to monitor changes in gene expression in cells. Already, there are chips with arrays representing all or many of the genes of the human and other species, such as rat, mouse, dog, monkey, honey bee, Arabidopis, Drosophila, and Escherichia coli.
3. DISEASE DIAGNOSIS:
DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.

4. DRUG DISCOVERY:
Drug discovery has evolved to become a large-scale and highly sophisticated endeavor. Microarray technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.
5. TOXICOLOGICAL RESEARCH:
Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). The simultaneous measurement of the entire human genome (thousands of genes) will facilitate the uncovering of specific gene expression patterns that are associated with disease.
6. COMPARATIVE GENOMIC HYBRIDIZATION (CGH) AND GENE COPY NUMBER
CGH is used as a molecular cytogenetic technique that permits quantitative analysis of gains and losses of whole or portions of chromosomes. A comprehensive high-resolution test for small chromosomal imbalances (microduplications or microdeletions) can now be performed in a single test. Investigators have developed a newer method that combines the principles of CGH with the use of microarrays. Instead of using metaphase chromosomes, this method—which is known as array CGH, or simply aCGH—uses slides arrayed with small segments of DNA as the targets for analysis. Microarray CGH works by comparing the levels of patient DNA to levels of a control (normal) DNA sample.In traditional CGH, DNAs from a "test" and a "reference" genome are differentially labeled with fluorophores and hybridized to normal metaphase chromosomes. In array CGH arrays of genomic BAC, P1, cosmid or cDNA clones are used for hybridization. CGH can not detect genomic changes involving <10–20 megabases. The use of microarray CGH overcomes many of these limitations, with improvement in resolution and dynamic range and improved throughput.










ROLE OF BIOINFORMATICS IN MICROARRAY TECHNOLOGY















Bioinformatics allows the researchers to tackle tasks easily by using Internet based tools without having to consider technical problems like local installation, maintenance or financial aspects. It aids in analysis of microarray technology by analyzing the expression levels, detecting anomalies, detecting SNP’s, finding GO terms, clustering, classification and then generating trees and processing fluorescent images.
TOOLS
1. Cryoscopy: Viewing a CGH (Comparative genomic hybridization) or expression data in a whole-genome context i.e. analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA. CGH will detect only unbalanced chromosomal changes. Structural chromosome aberrations such as balanced reciprocal translocations or inversions cannot be detected, as they do not change the copy number.

2. Array Viewer: Identification of statistically significant hybridization signals.

3. Cluster: Perform hierarchical clustering and self-organizing maps.

4. BAM array: Bayesian Analysis of Variance for Microarrays detect differentially expressed genes from multigroup microarray data.

5. Expression Profiler: Analysis & clustering of gene expression data.

6. TreeView: Graphically browse results of clustering and other analyses from Cluster.

















ROOM FOR IMPROVEMENT
AND
CONCLUSION













ROOM FOR IMPROVEMENT:
A lot of efforts have been made in the industry to improve the construction of the microarrays, as to advance the precision of the detection signals, increased speed of hybridization reactions, less usage of starting material and the cheaper availability of this technology.
"Microarrays will get better over time and a lot of that will be in content as we better understand which genes are important and, specifically, perhaps which splice variants are most important”, Says one of the scientists.
One of the improvements in the development phase is the usage of reflective substrates. It is speculated that printing microarrays on mirrors rather than glass improves the signal-to-noise ratio by as much as 1,000%.
CONCLUSION:
In recent decades, high-throughput scientific methods have been developed to optimize the study of large numbers of molecules, including DNA, proteins and metabolites. Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. DNA microarrays in particular have proved valuable in genomic research. They have been used to study gene expression patterns, to locate transcription factor binding sites, and to detect sequence mutations and deletions on a grand scale. However, DNA microarrays tell us only about the genes themselves and provide little information regarding the functions of the proteins they encode. Thus, protein microarrays were developed and were seen as high throughput approaches for the study of protein. On the other hand, tissue microarray is seen to provide a remarkable degree of standardization, speed, and cost efficiency for cancer analysis.












REFERENCES:

http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/basicbio.htm
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
http://www.chipscreen.com/articleFile/Microarrays-Tech%20and%20Application1.pdf
http://www.nature.com/nature/journal/v416/n6883/full/416885a.html
http://php.med.unsw.edu.au/cellbiology/index.php?title=Group_8_Project_-_Microarray
http://www.premierbiosoft.com/tech_notes/tissue-microarray.html
http://en.wikipedia.org/wiki/DNA_microarray
http://www.clinchem.org/cgi/content/full/47/8/1479
http://www.premierbiosoft.com/tech_notes/microarray.html
http://www.ncbi.nlm.nih.gov/geo/info/GEOHandoutFinal.pdf
http://www.sydneygenetics.com/Resources/WhatismicroarrayCGH/tabid/714/Default.aspx
http://www.ncbi.nlm.nih.gov/pubmed/12782098
http://chagall.med.cornell.edu/I2MT/MA-tools.pdf
http://discover.nci.nih.gov/tools.jsp
http://smd.stanford.edu/resources/restech.shtml
http://www.scq.ubc.ca/spot-your-genes-an-overview-of-the-microarray/
http://www.ambion.com/techlib/resources/microarray/basics1.html
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.fastol.com/~renkwitz/microarray_chips.htm