Selection of reference gene expression in a schizophrenia brain cohort

Abstract

Objective: In order to conduct postmortem human brain research into the neuropatho-logical basis of schizophrenia, it is critical to establish cohorts that are well-characterized and well-matched. The aim of the present study was therefore to determine if specimen characteristics including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs.

Methods: A matched cohort was selected of 74 subjects (schizophrenia/schizoaffective disorder, n = 37; controls, n = 37). Middle frontal gyrus tissue was pulverized, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using quantitative reverse transcription-polymerase chain reaction, nine housekeeper genes were measured and a geomean calculated per case in each diagnostic group.

Results: The RINs were very good (mean = 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH; two clinical variables, agonal state and duration of illness, did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. The results show that people with schizophrenia had significantly less PPIA and SDHA mRNA and tended to have less GUSB and B2M mRNA, suggesting that these control genes may not be good candidates for normalization.

Conclusions: In the present cohort <10% variability in RINs was detected and the diagnostic groups were well matched overall. The cohort was adequately powered (0.80-0.90) to detect mRNA differences (25%) due to disease. The study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.

Figure 1

Overall pH and RIN values similarly decreased with increasing age (both r=−0.27, p<0.020, n=74).

Figure 2

Average expression stability and optimal number of housekeeper genes for normalisation determined using the program geNorm. (A) Genes were ranked according to average expression stability. ACTB and GAPDH were the most stable genes (lowest M value), while GUSB was the least stable gene (highest M value). (B) Pairwise variation with the stepwise addition of more housekeeping genes shows that the use of the two most stable housekeeping genes will be sufficient for accurate normalisation as this satisfies the cutoff (0.15M) recommended by Vandesompele et al (2002).

Figure 3

(A) The calculated geomean(4) was comparatively similar in both diagnostic groups whether paired(t=−0.79, df=35, p=0.420) or unpaired (t=0.82 df=71, p=0.415); (B) The geomean(4) decreased with an increase in age (r=−0.28, p=0.018); (C) Overall pH in DLPFC tissue was positively correlated geomean(4) for controls (r=0.41, p=0.012) and patients (r=0.52, p=0.001); (D) Agonal state 1 versus 3 showed a significant decrease in the geomean(4) (p=0.031 by planned post-hoc LSD); (E) RIN values strongly correlated with the geomean(4) (r=0.57, p<0.0001); (E) PMI had no effect on the geomean(4) (r=0.006, p=0.96).

Figure 4

mRNA expression of housekeeping genes in control and schizophrenic DLPFC. mRNA expression was measured by quantitative RT-PCR and all cases expressed as a percentage of the control mean for that gene. (A) A significant reduction in expression of SDHA, and cyclophilin (PPIA) and strong trend in GUSB mRNAs was found in schizophrenia cases compared to controls (14.4%, p=0.016; 14.9%, p=0.015; and 12.4%, p=0.052 respectively by paired t-test). (B) mRNA expression levels for all remaining housekeeping genes that did not significantly differ between diagnostic groups, including the geomean(4) (all p>0.34).

Figure 5

The clinical variables that correlated with brain weight were daily CPZ (r=−0.34, p=0.038, n=37) (A) and duration of illness (B) (r=−0.35, p=0.031, n=37). Duration of illness negatively correlated with the geomean(4) shown in panel C (r=−0.35, p=0.034, n=36).