Microarrays

MICROARRAY

INTRODUCTION:

The term microarray was first introduced by Schena et al. in 1995 and the first genome of an eukaryotic species completely investigated (Saccharomyces cerevisiae) by a microarray was published in 1997 (Lashkari et al., 1997). In the last few years, further improvements were made especially when substituting the immobilized DNA-probes derived from clone-libraries by chemically synthesized oligonucleotides.

There are different names for the microarrays, like DNA/RNA Chips, BioChips or GeneChips. The array can be defined as an ordered collection of microspots, each spot containing a single defined species of a nucleic acid. The microarray technique is based on hybridization of nucleic acids. In this technique, sequence complementarity leads to the hybridization between two single-stranded nucleic acid molecules, one of which is immobilized on a matrix. There exist two variants of the chips: cDNA microarrays and oligonucleotide arrays. Although both the DNA and oligonucleotide chips can be used to analyze patterns of gene expression, fundamental differences exist between these methods. Two commonly used types of chips differ in the size of the arrayed nucleic acids. In cDNA microarrays, relatively long DNA molecules are immobilized by high-speed robots on a solid surface such as membranes, glass or silicon chips. Sample DNAs are amplified by the polymerase chain reaction (PCR) and usually are longer than 100 nucleotides. This type of arrays is used mostly for large-scale screening and expression studies.

The oligonucleotide arrays are fabricated either by in situ light-directed chemical synthesis or by conventional synthesis followed by immobilization on a glass substrate. Those with short nucleic acids (oligonucleotides up to 25 nucleotides) are useful for the detection of mutations and expression monitoring, gene discovery and mapping. In the procedure of genomic analysis, both types of microarrays are exposed to a labelled sample, hybridized, and complementary sequences are determined.

PRINCIPLE:

mRNA is an intermediary molecule which carries the genetic information from the cell nucleus to the cytoplasm for protein synthesis. Whenever some genes are expressed or are in their active state, many copies of mRNA corresponding to the particular genes are produced by a process called transcription. These mRNAs synthesize the corresponding protein by translation. So, indirectly by assessing the various mRNAs, we can assess the genetic information or the gene expression. This helps in the understanding of various processes behind every altered genetic expression. Thus, mRNA acts as a surrogate marker. Since mRNA is degraded easily, it is necessary to convert it into a more stable cDNA form. Labeling of cDNA is done by fluorochrome dyes Cy3 (green) and Cy5 (red). The principle behind microarrays is that complementary sequences will bind to each other.

The unknown DNA molecules are cut into fragments by restriction endonucleases; fluorescent markers are attached to these DNA fragments. These are then allowed to react with probes of the DNA chip. Then the target DNA fragments along with complementary sequences bind to the DNA probes. The remaining DNA fragments are washed away. The target DNA pieces can be identified by their fluorescence emission by passing a laser beam. A computer is used to record the pattern of fluorescence emission and DNA identification. This technique of employing DNA chips is very rapid, besides being sensitive and specific for the identification of several DNA fragments simultaneously.

Figure1: Principle of Microarray with reference to mRNA chip

PROCEDURE:

1.      Sample Collections: The samples can be a variety of organism. E.g. Two samples: cancerous human skin tissue and healthy human skin tissue

2.      Isolation of Nucleic acid: Every microarray study starts with the isolation of the respective targets (e.g. DNA or RNA). In principle, nucleic acids are isolated upon cell disruption by mechanical or enzymatic methods and precipitated at increased salt concentrations or ethanol.

mRNA Isolation: The extracted of RNAs using any methods (phenol-chloroform) is passed through the column containing beads with poly-T tails which bind the mRNA as it has a poly-A tail. All tRNA and rRNA are wshed out in this technique. Then it is rinsed with buffer to release the mRNA by disrupting the hybrid bonds with pH disturbance.

3.      Labelling: The next step is labelling of the molecules with fluorescent dyes; for that step various methods exist. Often the labelling step is done during the enzymatic amplification reaction at which fluorescently labelled nucleotides or primers are incorporated into the newly synthesized amplicons. Nowadays a broad range of fluorescent dyes with different absorption and excitation wavelengths are available. The different absorption and excitation maxima allow the combination of fluorescent dyes. In microarray analyses the fluorescent dyes Cy3 and Cy5 are widely used. cDNA labelled: The labelling mix contains poly-T primers, reverse transcriptase (to make cDNA) and fluorescently dyes nucleotides. The cyanine 3 (cy3-flouresce green) to the healthy cells and cyanine 5 (cy-5-fluoresce red) to the cancerous cells in this experiment.

4.      After purification of the labelled amplicons, these molecules are mixed with a hybridization buffer and are subsequently applied to the microarrays coating ssDNA probes and incubated overnight. After the hybridization procedure, unbound molecules have to be washed off before the detection can be done by laser-scanning with dye specific wavelengths.

5.      The detection step generates an image of the microarray, which is employed for raw data extraction. Thus, fluorescent intensities of each single spot of the microarrays are measured and written in a results file along with the spot coordinates and the specific “gene” identifier. The intensity of the generated signal depends on the number of molecules (targets), which have bound to the probe-molecules within one spot (also called feature). E.g. the higher intensity for red color indicates the up regulation for cancer cells and green intensities indicate the down regulation of gene expression for cancer cells.

6.      The last step in a microarray experiment is the bioinformatic analysis of the data of a single slide or data from many samples of distinct classes processed in parallel within one experiment.

Figure 2: The methods of microarrays

APPLICATIONS

·         The GeneChip technology may be employed in diagnostics (mutation detection), gene discovery, gene expression and mapping. It is used to measure expression levels of genes in bacteria, plant, yeast, animal and human samples.

·         At the present time, the main large-scale application of microarrays is comparative expression analysis.  The microarray technology provides the possibility to analyze the expression profiles for thousands of genes in parallel. Another application is the analysis of DNA variation on a genome-wide scale. Both of these applications have many common requirements. By hybridization with labelled mRNA, cDNA, arrayed PCR products or oligonucleotides on a substrate have been successfully used for monitoring transcript levels, single nucleotide polymorphism (SNP), or genomic variations between different strains.

·         One of the most significant applications of this technique is, as mentioned above, gene expression profiling on the whole genomic scale. For example, the expression levels of the genes in the Saccharomyces cerevisiae genome have been successfully determined with both the DNA and oligonucleotide microarray technology.

·         This technique has also been used to investigate physiological changes in human cells. DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome.

·         The microarray technology is a powerful yet economical tool for characterizing gene expression, regulation and will prove to be useful for strain improvement and bioprocess development. It may prove to be useful for strain development, process diagnosis, and process monitoring in bioreactors.

·         Information obtained from DNA chip analysis may enable researchers to determine the impact of a drug on a cell or group of cells, and consequently to determine the drug’s efficacy or toxicity. Knowledge of gene expression profiles can also help researchers to identify new drug targets.

·         The BioChip opens a new world of diagnostics based on genetics. This technology may be adequate to answer many medical questions. For example, gene expression profiles can be used for classification of tumors and for prognosis.

·         The technology finds increasing application in fundamental and applied research. The  major feature of this technique is that it allows one to perform a simultaneous analysis of a great number of DNA sequences.

·         The GeneChip technology is a new technique that undoubtedly will substantially increase the speed of molecular biology research.