Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain

Abstract

There are major concerns that specific agonal conditions, including coma and hypoxia, might affect ribonucleic acid (RNA) integrity in postmortem brain studies. We report that agonal factors significantly affect RNA integrity and have a major impact on gene expression profiles in microarrays. In contrast to agonal factors, gender, age, and postmortem factors have less effect on gene expression profiles. The Average Correlation Index is proposed as a method for evaluating RNA integrity on the basis of similarity of microarray profiles. Reducing the variance due to agonal factors is critical in investigating small but validated gene expression differences in messenger RNA levels between psychiatric patients and control subjects.

Figure 1

All 24 subjects with an agonal factor score (AFS) of 0 are highly correlated each other. (A) Pearson simple correlations of expression profile composed of 12,625 probe sets were calculated between all 780 pairs among 40 arrays from the anterior cingulate cortex. The 780 correlation coefficients are displayed in gray-scale (white, r > .94; gray, .90 < r < .94; black, r < .9). Almost all of the 24 subjects with an agonal factor score (AFS) of 0 are highly correlated (r > .94) with each other, regardless of mood disorder diagnoses. The 16 subjects with an AFS of 1 or more show lower correlations (r < .9) with most of the other subjects. This simple correlation matrix is used for calculating Average Correlation Index. The matrix is symmetrical around the diagonal line. (B) The distribution of the correlation coefficients calculated for subjects with no agonal factors (AFS = 0, open bars) is different than the distribution for subjects with agonal factors (AFS ≥ 1, filled bars). The individual data for the distribution of 276 correlation pairs among 24 subjects with an AFS of 0 and the correlation of 504 pairs between the 16 subjects with an AFS of 1 or more and all 40 subjects are shown in A. The mean correlation among subjects with an AFS of 0 was .968 (SD .014), whereas the correlation between subjects with an AFS of 1 or more and subjects with an AFS of 0 or subjects with an AFS of 1 or more were lower (mean .886, SD .055). There is a broad dispersion of correlations among subjects with an AFS of 1 or more, as shown in the range of correlation coefficients extending to .72. BPD, bipolar disorder; MDD, major depressive disorder.

Figure 2

Agonal factors and ribonucleic acid (RNA) degradation decrease Average Correlation Index (ACI). The ACI was calculated for the anterior cingulate cortex for 40 subjects and plotted on the y axis in the eight dot plots (A–H). The x axis shows the corresponding (A) agonal factor score (AFS); (B) brain tissue pH; (C) percentage of 18S and 28S ribosomal RNA to total RNA [%(18S+28S)]; (D) ratio of signal intensities for probe sets designed in 3′ and 5′ end region of glyceraldehyde phosphate dehydrogenase (GAPDH) gene (3′/5′ ratio of GAPDH; (E) the percentage of the total number of probe sets detected as present on the array (% Present Call); (F) coefficient of the RNA degradation slope from 5′ to 3′ ends of the averaged probe intensities calculated with the R-statistical program AffyRNAdeg (Degradation slope); (G) postmortem interval (PMI); and (H) freezer interval (FI). Average Correlation Index was significantly correlated with AFS and all of the RNA integrity indicators analyzed, whereas ACI showed no correlation with PMI and FI.

Figure 3

Average Correlation Index (ACI) of the microarray chips is a sensitive and specific indicator of agonal factors. The consistency of the ACI across three brain regions (anterior cingulate cortex, dorsolateral prefrontal cortex, and cerebellum) was examined for each subject. The ACI was averaged for each subject across the three brain regions and the result plotted by presence of agonal factors (agonal factor score [AFS] ≥ 1) or absence of agonal factors (AFS = 0). The vertical discrimination line is set to an ACI of .95. All subjects without an agonal factor were above this cut-off (n = 24), whereas 15 of 16 subjects with an agonal factor were below this ACI discrimination threshold. The ACI averaged across three brain regions was 100% sensitive to cases without agonal factors, with 93.7% specificity.