Hybridization interactions between probesets in short oligo microarrays lead to spurious correlations.
BACKGROUND
Microarrays measure the binding of nucleotide sequences to a set of sequence specific probes. This information is combined with annotation specifying the relationship between probes and targets and used to make inferences about transcript- and, ultimately, gene expression. In some situations, a probe is capable of hybridizing to more than one transcript, in others, multiple probes can target a single sequence. These 'multiply targeted' probes can result in non-independence between measured expression levels.


RESULTS
An analysis of these relationships for Affymetrix arrays considered both the extent and influence of exact matches between probe and transcript sequences. For the popular HGU133A array, approximately half of the probesets were found to interact in this way. Both real and simulated expression datasets were used to examine how these effects influenced the expression signal. It was found not only to lead to increased signal strength for the affected probesets, but the major effect is to significantly increase their correlation, even in situations when only a single probe from a probeset was involved. By building a network of probe-probeset-transcript relationships, it is possible to identify families of interacting probesets. More than 10% of the families contain members annotated to different genes or even different Unigene clusters. Within a family, a mixture of genuine biological and artefactual correlations can occur.


CONCLUSIONS
Multiple targeting is not only prevalent, but also significant. The ability of probesets to hybridize to more than one gene product can lead to false positives when analysing gene expression. Comprehensive annotation describing multiple targeting is required when interpreting array data.
Okoniewski
Michał J
MJ
Paterson Institute For Cancer Research, Christie Hospital site, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. MOkoniewski@PICR.man.ac.uk
Miller
Crispin J
CJ
D016477:Artifacts; D003198:Computer Simulation; D015342:DNA Probes / Q000235:genetics*; D003627:Data Interpretation, Statistical; D004867:Equipment Design; D004868:Equipment Failure; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods; D017403:In Situ Hybridization / Q000295:instrumentation* / Q000379:methods*; D008957:Models, Genetic; D015233:Models, Statistical; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013223:Statistics as Topic
D015342:DNA Probes