Abstract
BackgroundAffymetrix GeneChip technology enables the parallel observations of tens of thousands of genes. It is important that the probe set annotations are reliable so that biological inferences can be made about genes which undergo differential expression. Probe sets representing the same gene might be expected to show similar fold changes/z-scores, however this is in fact not the case.ResultsWe have made a case study of the mouse Surf4, chosen because it is a gene that was reported to be represented by the same eight probe sets on the MOE430A array by both Affymetrix and Bioconductor in early 2004. Only five of the probe sets actually detect Surf4 transcripts. Two of the probe sets detect splice variants of Surf2. We have also studied the expression changes of the eight probe sets in a public-domain microarray experiment. The transcripts for Surf4 are correlated in time, and similarly the transcripts for Surf2 are also correlated in time. However, the transcripts for Surf4 and Surf2 are not correlated. This proof of principle shows that observations of expression can be used to confirm, or otherwise, annotation discrepancies.We have also investigated groups of probe sets on the RAE230A array that are assigned to the same LocusID, but which show large variances in differential expression in any one of three different experiments on rat. The probe set groups with high variances are found to represent cases of alternative splicing, use of alternative poly(A) signals, or incorrect annotations.ConclusionOur results indicate that some probe sets should not be considered as unique measures of transcription, because the individual probes map to more than one transcript dependent upon the biological condition. Our results highlight the need for care when assessing whether groups of probe sets all measure the same transcript.
Highlights
Affymetrix GeneChip technology enables the parallel observations of tens of thousands of genes
Our results indicate that some probe sets should not be considered as unique measures of transcription, because the individual probes map to more than one transcript dependent upon the biological condition
Only five of the probe sets were correctly assigned to the reverse strand of chromosome 2, whereas two probe sets align to the forward strand of chromosome 2 and one probe set aligns to chromosome 19
Summary
Affymetrix GeneChip technology enables the parallel observations of tens of thousands of genes. One of the most widely used microarray platforms is the Affymetrix GeneChip. A GeneChip consists of a quartz wafer to which are attached some 500,000 different 25mer deoxyoligonucleotides, which are known as probes [1]. Gene expression is measured by extracting mRNA from the cells or tissues of interest and hybridising the mRNA sample to the 25-mer probes on the microarray. Each expressed transcript is represented on an array by a series of probe pairs known as a probe set [1]. Each probe set on the Affymetrix MOE430A and RAE230A arrays consists of 11 probe pairs, and is given a unique identifier consisting of a seven digit number, followed by the optional characters _s, _a, or _x, and ending in _at [2,3]
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