Affymetrix GeneChip ® Probe Arrays
The single extract technique describedpreviously is used to hybridise the arrays made by Affymetrix. The arrays comprise oligonucleotides fixed to the surface of the chip using a combination of photolithography and combinatorial chemistry. These oligonucleotides are known as probes, because they probe or interrogate the sample, and the chips themselves are known as GeneChip probe arrays. To hybridise the array, cDNA is synthesised from total RNA isolated from the samples to be analysed. An in vitro transcription reaction is then performed to produce cRNA labelled with biotin, after which the cRNA is fragmented before hybridisation. This labelled nucleic acid is known as the target. Following a 16-hour hybridisation, the sample is washed, stained and scanned.
Affymetrix Microarray Software
The Affymetrix software suite defines specific areas of the probe array known as probe cells, each of which contains oligonucleotides derived from a specific gene sequence. For each gene, 11 (ATH1) or 16 (AtGenome1) perfect or mismatch probe pair sets of 25bp oligomers are contained adjacent to each other in a probe cell. The mismatch probes have a single mismatch at position 13 of the strand; this enables any non-specific hybridisation to the probe cell sequence to be measured. The Affymetrix software is used to calculate gene expression data, based upon average differences seen in the intensity values of each probe cell. The resulting image file is stored as a .dat file. The .dat file can be analysed against a large range of internal controls (AFFYMETRIX analysis handbook). A .cel image file is created for each .dat file. In all cases I was provided with both .dat and .cel files from the laboratories performing the hybridisation and scanning of the Affymetrix GeneChips.
Affymetrix GeneChip Expression Analysis
Analysis of expression data includes the process of scaling. In this process the average intensity value is scaled relative to a common target intensity value enabling data from different chips to be compared. For example, if the average intensity value of chip 1 is 200 and that of chip 2 is 300, without scaling the two chips to a common value, chip 2 is always likely to give higher expression levels than chip 1.
The differences in initial intensity values are usually due to assay variables such as pipetting errors that are independent of the relative transcript concentration. All of the chip sets that were used in the experiments described in this chapter were scaled using a scaling factor of 250, as recommended by Affymetrix for the AtGenome1 GeneChip. Single array analysis is then performed. This process allows the creation of gene expression data matrices. These are used for subsequent classification of changes in gene transcription levels, and for procedures such as gene clustering.
The data from single array analysis is stored as a .chp file, which is created from the .cel file. Single array analysis creates a signal value for each probe cell that is a measure of the abundance of the transcript. This analysis generates a detection p-value, which is used to give a detection call for each probe.