High-resolution quantification of specific mRNA levels in human brain autopsies and biopsies

Abstract

Quantification of mRNA levels in human cortical brain biopsies and autopsies was performed using a fluorogenic 5' nuclease assay. The reproducibility of the assay using replica plates was 97%-99%. Relative quantities of mRNA from 16 different genes were evaluated using a statistical approach based on ANCOVA analysis. Comparison of the relative mRNA levels between two groups of samples with different time postmortem revealed unchanged relative expression levels for most genes. Only CYP26A1 mRNA levels showed a significant decrease with prolonged time postmortem (p = 0.00004). Also, there was a general decrease in measured mRNA levels for all genes in autopsies compared to biopsies; however, on comparing mRNA levels after adjusting with reference genes, no significant differences were found between mRNA levels in autopsies and biopsies. This observation indicates that studies of postmortem material can be performed to reveal the relative in vivo mRNA levels of genes. Power calculations were done to determine the number of individuals necessary to detect differences in mRNA levels of 1.5-fold to tenfold using the strategy described here. This analysis showed that samples from at least 50 individuals per group, patients and controls, are required for high-resolution ( approximately twofold changes) differential expression screenings in the human brain. Experiments done on ten individuals per group will result in a resolution of approximately fivefold changes in expression levels. In general, the sensitivity and resolution of any differential expression study will depend on the sample size used and the between-individual variability of the genes analyzed.

Figure 1

Experimental strategy. Three brain autopsies (or biopsies) were extracted from each individual. These triplicates were analyzed and processed in parallel. PolyA + RNA prepared from brain was reverse transcribed and cDNA was quantified using a real-time PCR method in which a labeled probe is degraded during PCR by Taq polymerase exonuclease activity.

Figure 2

Correlation studies. Identical replica plates containing 81 cDNA samples obtained from brain. The samples were extracted in triplicate from 27 individuals. The replica plates were used to measure twice the expression of ACTB (A) or D5 (C). Two reference genes (ACTB and GAPD) show a correlation of 96% (B). A gene with high between-individual variability (D5) showed a low correlation to actin expression levels (D). Axes show logarithm of measured number of cDNA copies.

Figure 3

Significant differences in relative expression levels for sex, age, and time postmortem. ANCOVA analysis compare means of measured cDNA copies adjusted with the number of cDNA copies for ACTB and GAPD. Significant differences found in the analysis of 5-HT-1E, CYP26A1, and CYP1A1, as shown in Table 1, are visualized in the differences between the regression lines of the two groups compared. Axes show logarithm of measured number of cDNA copies.

Figure 4

Comparison of expression levels between human brain autopsies and biopsies. Absolute expression levels are decreased in autopsies compared to biopsies; however, the differences between the two groups were not significant (no P-value <0.0005) after adjustment with reference genes. The level of significance was selected after a Bonferroni correction for 100 independent tests performed. The boxes show the interval between − 1 and + 1 standard error, and the whiskers show 95% confidence interval. Axes show logarithm of measured number of cDNA copies. The gray line separates two sets of genes measured with two different sets of replica plates.

Figure 5

Distribution of adjusted expression levels in individuals. The expression values (number of cDNA copies amplified) for the 3 samples extracted from each individual were used to calculate adjusted means for each individual as described in the Methods section. The boxes show standard deviations, the dots indicate the means for each gene, and whiskers mark the 95% confidence intervals. Mean (log) expression levels of each individual have been adjusted with reference genes ACTB and GAPD. The adjusted values of expression for each individual and each gene are available at our website (see http://www.genpat.uu.se/psge/psgecastensson.html).