Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples

Abstract

Background: The Affymetrix GeneChip® system is a commonly used platform for microarray analysis but the technology is inherently expensive. Unfortunately, changes in experimental planning and execution, such as the unavailability of previously anticipated samples or a shift in research focus, may render significant numbers of pre-purchased GeneChip® microarrays unprocessed before their manufacturer's expiration dates. Researchers and microarray core facilities wonder whether expired microarrays are still useful for gene expression analysis. In addition, it was not clear whether the two human reference RNA samples established by the MAQC project in 2005 still maintained their transcriptome integrity over a period of four years. Experiments were conducted to answer these questions.

Results: Microarray data were generated in 2009 in three replicates for each of the two MAQC samples with either expired Affymetrix U133A or unexpired U133Plus2 microarrays. These results were compared with data obtained in 2005 on the U133Plus2 microarray. The percentage of overlap between the lists of differentially expressed genes (DEGs) from U133Plus2 microarray data generated in 2009 and in 2005 was 97.44%. While there was some degree of fold change compression in the expired U133A microarrays, the percentage of overlap between the lists of DEGs from the expired and unexpired microarrays was as high as 96.99%. Moreover, the microarray data generated using the expired U133A microarrays in 2009 were highly concordant with microarray and TaqMan® data generated by the MAQC project in 2005.

Conclusions: Our results demonstrated that microarray data generated using U133A microarrays, which were more than four years past the manufacturer's expiration date, were highly specific and consistent with those from unexpired microarrays in identifying DEGs despite some appreciable fold change compression and decrease in sensitivity. Our data also suggested that the MAQC reference RNA samples, stored at -80°C, were stable over a time frame of at least four years.

Figure 1

The workflow of study design and data analysis New gene expression data were generated in 2009 with expired U133A microarrays and unexpired U133Plus2 microarrays using the same MAQC reference RNA samples and compared to the microarray and TaqMan^® gene expression data generated in 2005 by the MAQC project.

Figure 2

Comparisons of the log₂ fold changes detected in 2009 and in 2005 using the same type of U133Plus2 microarrays (a) all 8,550 common genes; (b) the 7,069 genes with p < 0.05 in either 2009 or 2005; and (c) the 5,880 genes with p < 0.05 in both 2009 and 2005. Fold changes were generated by comparing sample B to sample A, i.e., B/A. The blue and green dots indicated the up- and down-regulated genes, respectively. The red dots were the genes with reverse regulation directionalities in 2009 and in 2005. Under the three scenarios (a, b, and c), the overlap of differentially expressed genes between 2009 and 2005 is 91.37%, 97.44%, and 99.78%, respectively, suggesting a high degree of stability of the two MAQC reference RNA samples.

Figure 3

Correlations of log₂ intensities generated in 2009 among the replicates of samples A and B (a) expired U133A microarrays and (b) unexpired U133Plus2 microarrays. Each scatterplot represents the comparison of log₂ intensities of 8,550 common genes from two hybridizations.

Figure 4

Comparisons of log₂ fold changes detected with expired U133A and unexpired U133Plus2 microarrays (a) all 8,550 common genes; (b) the 6,835 genes with p < 0.05 in either expired U133A microarrays (2009) or unexpired U133Plus2 microarrays (2009); and (c) the 5,120 genes with p < 0.05 in both expired U133A (2009) and unexpired U133Plus2 (2009) microarrays. Fold changes were generated by comparing sample B to sample A, i.e., B/A. The blue and green dots indicated the up- and down-regulated genes, respectively. The red dots were the genes with reverse regulation directionalities. Under the three scenarios (a, b, and c), the overlap of differentially expressed genes between expired U133A (2009) and unexpired U133Plus2 (2009) microarrays is 89.94%, 96.99%, and 99.98%, respectively, indicating a high degree of consistency between differential gene expression data generated from expired U133A and unexpired U133Plus2 microarrays. Note that the fold changes measured with expired U133A microarrays exhibited some degree of compression (i.e., with smaller absolute values) when compared to those obtained with unexpired U133Plus2 microarrays.

Figure 5

Concordances of different microarrays in terms of percentage of overlapping genes (POG) The x-axis represents the number of genes (2L) selected as differentially expressed, and the y-axis represents the overlap (%) of two gene lists selected from the two experiments under comparison. All of the genes in the two compared experiments were first filtered with a p < 0.05 cutoff and then ranked by fold changes in up- and down directions. Next, the top L genes with the largest fold changes were selected as differentially expressed from each direction and the DEG list containing 2L genes from each experiment was used for cross-experiment comparisons. L was increased from one to the lower number of genes in both regulation directions with a step of one. The red crosses represent the reference line, i.e., the mean POG of DEG lists in four comparisons of microarray reference data groups in MAQC project: ABI, AG1, ILM, and GEH versus AFX. The dashed and dotted green and blue markers showed the lower limit and the upper limit of the 95% confidence intervals, respectively, for the reference line.

Figure 6

Concordance between TaqMan^® assays and microarray data in terms of percentage of overlapping genes (POG) The x-axis represents the number of genes (2L) selected as differentially expressed, and the y-axis represents the overlap (%) of two gene lists selected from the two experiments under comparison. See notes under Figure 5 for more information.

Figure 7

Distribution of the log₂ intensities of 8,550 common genes Sample A hybridized on expired U133A (a) and unexpired U133Plus2 (b) microarrays; Sample B hybridized on expired U133A (c) and unexpired U133Plus2 (d) microarrays. For each gene, the log₂ intensities of the three technical replicates were averaged. Based on microarray data generated in 2009.

Figure 8

Percentage of Present calls based on 8,550 common genes There is a significant difference in the percentage of Present calls between unexpired U133Plus2 microarrays (73.6%) and expired U133A microarrays (64.5%). Based on microarray data generated in 2009.