The purified RNA is analysed for quality (by capillary electrophoresis) and quantity (for example, by using a NanoDrop or NanoPhotometer spectrometer). If the material is of acceptable quality and sufficient quantity is present (e.g., >1μg, although the required amount varies by microarray platform), the experiment can proceed.
The labelled product is generated via reverse transcription and sometimes with an optional PCR amplification. The RNA is reverse transcribed with either polyT primers (which amplify only mRNA) or random primers (which amplify all RNA, most of which is rRNA); miRNA microarrays ligate an oligonucleotide to the purified small RNA (isolated with a fractionator), which is then reverse transcribed and amplified. The label is added either during the reverse transcription step, or following amplification if it is performed. The sense labelling is dependent on the microarray; e.g. if the label is added with the RT mix, the cDNA is antisense and the microarray probe is sense, except in the case of negative controls. The label is typically fluorescent; only one machine uses radiolabels. The labelling can be direct (not used) or indirect (requires a coupling stage). For two-channel arrays, the coupling stage occurs before hybridization, using aminoallyluridinetriphosphate (aminoallyl-UTP, or aaUTP) and NHS amino-reactive dyes (such as cyanine dyes); for single-channel arrays, the coupling stage occurs after hybridization, using biotin and labelled streptavin. The modified nucleotides (usually in a ratio of 1 aaUTP: 4 TTP (thymidine triphosphate)) are added enzymatically in a low ratio to normal nucleotides, typically resulting in 1 every 60 bases. The aaDNA is then purified with a column (using a phosphate buffer solution, as Tris contains amine groups). The aminoallyl group is an amine group on a long linker attached to the nucleobase, which reacts with a reactive dye. A form of replicate known as a dye flipcan be performed to remove any dye effects in two-channel experiments; for a dye flip, a second slide is used, with the labels swapped (the sample that was labeled with Cy3 in the first slide is labeled with Cy5, and vice versa). In this example, aminoallyl-UTP is present in the reverse-transcribed mixture.
The mixture is denatured and added to the pinholes of the microarray. The holes are sealed and the microarray hybridized, either in a hyb oven, where the microarray is mixed by rotation, or in a mixer, where the microarray is mixed by alternating pressure at the pinholes.
After an overnight hybridization, all nonspecific binding is washed off (SDS and SSC).
The microarray is dried and scanned by a machine that uses a laser to excite the dye and measures the emission levels with a detector.
The image is gridded with a template and the intensities of each feature (composed of several pixels) is quantified.
The raw data is normalized; the simplest normalization method is to subtract background intensity and scale so that the total intensities of the features of the two channels are equal, or to use the intensity of a reference gene to calculate the t-value for all of the intensities. More sophisticated methods include z-ratio, loess and lowess regression and RMA (robust multichip analysis) for Affymetrix chips (single-channel, silicon chip, in situ synthesised short oligonucleotides).