Validation of commonly used reference genes for sleep-related gene expression studies

Autor(es)

Lee, Kil S. ^[1] ; Alvarenga, Tathiana A. ^[2] ; Guindalini, Camila ^[2] ; Andersen, Monica L. ^[2] ; Castro, Rosa M. R. P. S. ^[2] ; Tufik, Sergio ^[2]

Afiliação do(s) autor(es)

^[1] AFIP, BR-04024002 Sao Paulo - Brazil

^[2] Univ Fed Sao Paulo, Dept Psychobiol, BR-04024002 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BMC MOLECULAR BIOLOGY; v. 10, MAY 15 2009.
• Citações Web of Science: 15

Resumo

Background: Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD) on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine guanine phosphoribosyl transferase (HPRT). Results: Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF) and glycerol-3-phosphate dehydrogenase1 (GPD1) was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. Conclusion: This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results. (AU)

• Processo FAPESP: 06/58274-5 - Influencia da restricao alimentar no comportamento sexual de ratos machos.
• Beneficiário: Tathiana Aparecida Fernandes de Alvarenga
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 98/14303-3 - Center for Sleep Studies
• Beneficiário: Sergio Tufik
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs