Microarrays exploit the preferential binding of complementary single-stranded nucleic acid sequences. A microarray is typically a glass slide, on to which DNA molecules are attached at fixed locations (spots). There may be tens of thousands of spots on an array, each containing a huge number of identical DNA molecules (or fragments of identical molecules), of lengths from twenty to hundreds of nucleotides. (According to quick napkin calculations by Wilhelm Ansorge and John Quackenbush in Schnookeloch in Heidelberg on 4 October 2001, the number of DNA molecules in a microarray spot is 107-108). For gene expression studies, each of these molecules ideally should identify one gene or one exon in the genome, however, in practice this is not always so simple and may not even be generally possible due to families of similar genes in a genome.
Microarrays that contain all of the approximate 6000 genes of the yeast genome have been available since 1997. The spots are either printed on the microarrays by a robot, or synthesised by photolithography (similarly as in computer chip productions) or by ink-jet printing. The spot diameter is of the order of 0.1 mm, for some microarray types can be even smaller.
There are different ways how microarrays can be used to measure the gene expression levels. One of the most popular microarray applications allows the comparison of gene expression levels in two different samples, e.g., the same cell type in a healthy and diseased state. This is called cDNA microarray. An other array technique is oligo arrays.
- cDNA Microarray
In the preparation of a cDNA microarray, the total mRNA from the cells in two different conditions is extracted and reverse transcription PCR (RT-PCR) is used to convert the RNA transcripts into cDNA. The cDNAs are usually composed of 500 -2000 basepairs long. The complete pool of cDNA is representative of transcriptional events in the tissue source of the RNA. The genes that were being actively transcribed in the sample will have mRNA copies that should have been first purified and then copied into cDNA during the RT-PCR step. The reverse transcription event for the control and experimental mRNA are identical in every step except one, and it is this step that enables differential gene expression to be determined. Nucleotides labelled with a green fluorescent dye Cy3 are incorporated into the control cDNA, while nucleotides labelled with a red fluorescent dye Cy5 are incorporated into the experimental DNA. After preparation, both probes are mixed and allowed to hybridise to the glass slide. Excess hybridisation buffer is washed off following an overnight incubation, and the slides are then ready to be scanned. Labelled gene products from the extracts hybridise to their complementary sequences in the spots due to the preferential binding - complementary single stranded nucleic acid sequences tend to attract to each other and the longer the complementary parts, the stronger the attraction.
- Oligonucleotide Microarray
The physical chemistry of hybridisation is oligonucleotide microarrays is clearly different from that of cDNA microarrays. Oligonucleotides range in size from 10-25 bases. So, the DNA fragments in the spots are much smaller than cDNA fragments are. Oligonucleotide microarrays are used to detect point mutations (the missing, adding or changing of a single base) in a known DNA sequence. Single base mismatches do have much more influence on binding to an oligonucleotide sequence compared to cDNA. For example, a small genome can be synthesized on a chip as a set of thousands of 20 bp long fragments. When a single basepair match exists, the fluorescence intensity decreases significant. This technique gives possibilities to find most of the point mutations in a known DNA sequence.
Data quantification
The dyes enable the amount of sample bound to a spot to be measured by the level of fluorescence emitted when a laser excites it. If the RNA from the sample in condition 1 is in abundance, the spot will be green, if the RNA from the sample in condition 2 is in abundance, it will be red. If both are equal, the spot will be yellow, while if neither are present it will not fluoresce and appear black. Thus, from the fluorescence intensities and colours for each spot, the relative expression levels of the genes in both samples can be estimated.
The raw data that are produced from microarray experiments are the hybridised microarray images. To obtain information about gene expression levels, these images should be analysed, each spot on the array identified, its intensity measured and compared to the background. This is called image quantification and is done by image analysis software. To obtain the final gene expression matrix from spot quantification's, all the quantities related to some gene (either on the same array or on arrays measuring the same conditions in repeated experiments) have to be combined and the entire matrix has to be scaled to make different arrays comparable.
Microarrays are already producing massive amounts of data. These data, like genome sequence data, can help us to gain insights into underlying biological processes only if they are carefully recorded and stored in databases, where they can be queried, compared and analysed by different computer software programs. The EBI as well as the NCBI are establishing a public repository for microarray gene expression data analogous to banks for DNA sequence data.
Microarray is fundamentally a technique to identify complete gene expression profiles in selected tissues. Microarray experiments can give false positive and false negative results. Additional means of analysing gene expression (Northern blotting or RNAse protection assays) must be used to control microarray conclusion.
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