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

Hospital Universitari Psiquiàtric Institut Pere Mata, IISPV. Universitat Rovira i Virgili, C/ Sant Llorenç 21, 43201 Reus, Spain.
Journal of Psychiatric Research (Impact Factor: 3.96). 06/2011; 45(11):1411-8. DOI: 10.1016/j.jpsychires.2011.06.001
Source: PubMed

ABSTRACT 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.
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.
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.
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).
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.

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Available from: Lourdes Martorell, Jun 16, 2014
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    • "Since it has been reported that GAPDH expression is altered in neurodegenerative diseases [11], we evaluated several endogenous controls by qRT-PCR in the frontal cortex regions and RPLP0 was chosen because this transcript presented overall low Ct values and lowest Ct variation (unpublished data). In addition, RPLP0 has been used as a stable reference gene in previous studies [1, 16, 31]. "
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    • "Suitable housekeeping genes as an internal control under certain defined conditions have been reported in previous studies. Rho et al.7 have reported that RPL29 only or the combination of RPL29 and B2M was most appropriate for stomach tissues; B2M only or the combination of B2M and GAPDH for stomach cancer cell lines;7 ACTB and SDHA, for the gene expression analysis of breast cancer among typical reference genes;8 and GAPDH, RPS17, RPL30, RPLP0, and TFRC, in studies on post-mortem brain samples from patients with major psychiatric disorders.9 According to gene expression studies of mice myocardial infarction models, HPRT, RPL13A, and TPT1 are the appropriate reference genes;10 GUSB, PPIA, and TBP, are used for serous ovarian cancer;11 and EEF1A1 only or the combination of EEFA1 and GAPDH, are commonly used for cervical cancer.12 "
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