RT-qPCR study on post-mortem brain samples from patients with major psychiatric disorders: reference genes and specimen characteristics.

BACKGROUND: Gene expression studies conducted in post-mortem human brain samples have the potential to identify relevant genes implicated in psychiatric disorders. Although reverse transcription quantitative real-time PCR (RT-qPCR) has emerged as the method of choice for specific gene expression studies, it requires the use of stable reference genes, and it is necessary to control for pre- and post-mortem factors to obtain reliable data.
OBJECTIVE: The aim of this study was to identify suitable reference genes and specimen characteristics that can be taken into account when comparing mRNA expression data between post-mortem brain specimens from psychiatric patients and controls.
METHOD: We used a selection of suitably matched occipital cortex specimens from subjects in each of the following groups: schizophrenia (N = 15), bipolar disorder (N = 13), major depressive disorder (N = 15), and control (N = 15). Quantitative and qualitative RNA analyses were performed prior to RT-qPCR and gene expression stability was evaluated with geNorm and NormFinder.
RESULTS: We identified GAPDH, RPS17, RPL30, RPLP0, and TFRC as potential reference genes from a sample plate containing 32 candidates commonly used as reference genes. Further analyses of these 5 genes highlighted that 1) they are suitable reference genes for RT-qPCR studies in these post-mortem brain samples from psychiatric patients, and 2) the RNA quality index is highly correlated with gene expression values (r = -0.681, p < 0.0001).
CONCLUSIONS: In addition to controlling for pre- and post-mortem factors and selecting stable reference genes for normalization, sample sets should be matched with regard to RNA quality.