DNA microarray principle
DNA microarrays allow for rapid measurement and visualisation of differential expression between genes at the whole genome scale. If technique implementation is quite complicated, its principle is very easy. Here are described the major steps involved in this process:
Microarray production process:
DNA fragments amplified by PCR technique are spotted on a microscopic glass slide coated with polylysine prior to spotting process. The polylysine coating goal is to ensure DNA fixation through electrostatic interactions. PCR fragments are in our case the expressed part (ORF) of the 6200 Saccharomyces cerevisae genes (baker yeast). Slide preparation is achieved by blocking the polylysine not fixed to DNA in order to avoid target binding. Prior to hybridisation, DNA is denatured to obtained a single strand DNA on the microarray, this will allow the probe to bind to the complementary strand from the target. Apart from glass slide microarray other types of chips exist:
RNA are extracted from two yeast cultures from which we want to compare expression level. Messengers RNA are then transformed in cDNA by reverse transcription. On this stage, DNA from the first culture with a green dye, whereas DNA from the second culture is labelled with a red dye.
Green labelled cDNA and red labelled ones are mixed together (call the target) and put on the matrix of spotted single strand DNA (call the probe). The chip is then incubated one night at 60 degrees. At this temperature, a DNA strand that encounter the complementary strand and match together to create a double strand DNA. The fluorescent DNA will then hybridise on the spotted ones.
A laser excites each spot and the fluorescent emission gather through a photo-multiplicator (PMT) coupled to a confocal microscope. We obtained two images where grey scales represent fluorescent intensities read. If we replace grey scales by green scales for the first image and red scales for the second one, we obtained by superimposing the two images one image composed of spots going from green ones (where only DNA from the first condition is fixed) to red (where only DNA from the second condition is fixed) passing through the yellow colour (where DNA from the two conditions are fixed on equal amount).
We have now two microarray images from which we have to calculate the number of DNA molecules in each experimental condition. To dos o, we measure the signal amount in the green dye emission wavelength and the signal amount in the red dye emission wavelength. Then we normalise these amount according to various parameters (yeast amount in each culture condition, emission power of each dye, ). We suppose that the amount of fluorescent DNA fixed is proportional to the mRNA amount present in each cell at the beginning and we calculate the red/green fluorescence ratio. If this ratio is greater than 1 (red on the image), the gene expression is greater in the second experimental condition, if this ration is smaller than 1 (green on the image), the gene expression is greater in the first condition. We can visualize these differences in expression using software as the one developed in the laboratory call ArrayPlot (cf below image). This software allows from the intensities list of spot to display the red intensities of each spot as a function of the green intensities.
Expression profile clustering:
Then we can try to gather genes that share the same expression profile on several experiments. This clustering can be done gradually as for phylogenetic analysis, which consist in calculating similarity criteria between expression profiles and gather the most similar ones. We can also use more complex techniques as principal component analysis or neuronal networks.
At the end hierarchical clustering is usually displayed as a matrix where each column represent one experiment and each row a gene. Ratios are displayed thanks to a colour scale going from green (repressed genes) to red (induced genes).
Stéphane LE CROM and Philippe MARC
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