Improved precision and accuracy for microarrays using updated probe set definitions

Abstract

Background: Microarrays enable high throughput detection of transcript expression levels. Different investigators have recently introduced updated probe set definitions to more accurately map probes to our current knowledge of genes and transcripts.

Results: We demonstrate that updated probe set definitions provide both better precision and accuracy in probe set estimates compared to the original Affymetrix definitions. We show that the improved precision mainly depends on the increased number of probes that are integrated into each probe set, but we also demonstrate an improvement when the same number of probes is used.

Conclusion: Updated probe set definitions does not only offer expression levels that are more accurately associated to genes and transcripts but also improvements in the estimated transcript expression levels. These results give support for the use of updated probe set definitions for analysis and meta-analysis of microarray data.

Figure 1

Improved precision using updated probe set definitions. (A) We compared the precision in microarray experiments when using the updated probe set definitions in comparison with the original probe set definition (netAffx). We measured the precision as the Pearson correlation between two log2 ratios of expression levels generated by comparing duplicate sample-vs-control comparisons. The measurement indicates how well a fold change can be reproduced. Data from five different data sets generated using identical samples (data normalized using RMA) were averaged (mean) [see Additional file 1] for similar figures for each laboratory independently). The x-axis shows the number of probes integrated into each probe set. We grouped the number of probes into intervals of three in order to obtain a sufficiently large number of probe sets to get a robust estimate of each data point. The y-axis shows the mean correlation/precision across the five labs for each interval. (B) Visualizing the number of probe sets in each interval between the different probe set definitions.

Figure 2

Improved accuracy using updated probe set definitions. The accuracy between measured log2 ratios using RT-PCR and microarrays were compared for microarray measurements using the original (NetAffx) and updated probe set definitions. The accuracy was obtained by calculating the slope from a linear regression comparing the obtained log2 ratios from the microarray to those estimates obtained using RT-PCR, where the microarray estimates were obtained using the different probe set definitions. A slope of 1 would indicate perfect accuracy. The mean accuracy across five different labs is plotted together with the standard deviations.