As a prerequisite to this article you must understand what genes, DNA, proteins and mRNA are. You can find our short introduction to these concepts here.
DNA microarrays are a high throughput technology used to measure the expression levels of thousands of genes, in some cases all of the genes in a genome, simultaneously. The fundamental idea behind most microarrays is to exploit complementary base pairing (see previous section) to measure the amount of the different types of mRNA molecules in a cell, thus indirectly measuring the expression levels of the genes that are responsible for the synthesis of those particular mRNA molecules.
The spots on a microarray contain single stranded DNA oligonucleotides called probes. Each of these spots will contain DNA which is of a complementary sequence to the specific mRNA molecule that corresponds to the gene that it is targeting. An mRNA molecule which is complementary to the probe in question, should hybridise to that probe, forming a strong mRNA-DNA bond. These mRNA molecules will have previously been labeled with fluorescent dye, which means that the amount of hybridisation that has taken place can be measured by the level of fluorescence of the dye, which is examined with a scanner. This scanner then outputs a text file for each array, which contains the relevant data pertaining to that array, such as the level of fluorescence of each spot and the level of background noise. It is these text files which are subsequently computationally analysed. In theory, a spot with brighter fluorescence means that more mRNA has hybridised, which in turn infers that more mRNA was present in sample extracted from the original cell and that the gene represented by this spot is experiencing a higher level of expression.
The types of DNA microarrays most widely used today can be broadly divided into two categories, cDNA arrays and oligonucleotide arrays.
The Affymetrix GeneChip
The GeneChip, which is manufactured by Affymetrix, is an oligonucleotide array and is the most commonly used type of DNA microarray. They differ slightly in operation from other kinds of arrays. Each array will contain hundreds of thousands of probe spots and each of these spots will in turn contain millions of copies of an individual 25 base long DNA oligonucleotide.
Each gene that is being targeted is represented by typically (but not necessarily always) by 11 pairs of these probes. This set of probes contains 11 perfect match (PM) probes, which are exactly complementary to the DNA sequence of a subset of 25 bases of the target gene. Each PM probe has a corresponding mismatch probe (MM), which contains the same 25 base long sequence as the PM probe, except for the fact that the middle base, or the 13th base in the chain, is substituted for the complement of the 13th base of its corresponding PM probe; so for example, a G in the 13th base of a PM probe will be replaced with a C in the MM probe. This is meant to give an estimate of non-specific binding, which occurs when mRNA that is not targeted binds to a PM probe.
cDNA microarrays differ from Affymetrix arrays in that each spot corresponds entirely to a specific gene. Sometimes duplicate spots will target the same gene, but these spots are exact copies of each other. The probes are of varying length but are generally hundreds of bases long. Instead of mRNA levels being directly measured, these arrays measure complementary DNA (cDNA), because this is more stable molecule than mRNA at these large sizes. mRNA from the original sample is reverse transcribed in a laboratory to create an equivalent number of the more stable cDNA molecules which are then hybridised to the microarray. These cDNA molecules are usually more than 500 bases long. Each of the probes contained on the spots on the microarary will be complementary to a cDNA molecule that represents a given gene. Thus, the measure of how much cDNA binds to its corresponding spot gives an accurate measure of the expression level of the gene in question, assuming that nothing has gone wrong.
Also instead of expression levels of an individual sample being measured directly, two separate samples are hybridised to the same array at the one time. One of these samples is generally a control sample, while the other one is a sample of interest such as tumour tissue. Each of these samples is labeled with a particular dye; either a red-fluorescent dye, Cyanine 5 or Cy5, or a green-fluorescent dye, Cyanine 3 or Cy3. When the array is read by the scanner, the differential expression level of a given gene is measured by the difference in intensity level between the red and green channel, at the spot that corresponds to the gene in question.
cDNA microarrays are initially read by a scanner, which produces a TIFF image of the array. These images are then interpreted by one of a number of image analysis software packages, all of which output data in slightly different formats. This system supports analysis of data from the major platforms, including Spotfire, GenePix, BlueFuse and Agilent.
Microarrays can also be used for detection of miRNA expression levels. miRNA are short RNA molecules, generally about 22 nucleotides in length. They are encoded in genes but are not translated into proteins; instead, these molecules down regulate the expression of certain genes. They achieve this by being complementary to specific mRNA molecules created in a cell. The miRNA molecules bind to the complementary sections of these mRNAs and stop them from being converted into proteins.
Exiqon manufacture microarrays for detection of miRNA expression. The spots on these microarrays consist of Locked Nucleic Acid (LNA) probes. LNA is a modified RNA nucleotide that, because of the short length of miRNA molecules, forms a more stable bond with miRNA than standard DNA probes meaning that accuracy of measurements is increased. The miRNA molecules that are being targeted will bind to its complementary LNA probe.
Other than this the processes of labeling the sample with a fluorescent dye, hybridising the sample to the array and reading the hybridisation levels with a scanner are similar to those of other arrays.