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

doi:10.1186/1471-2199-10-45

BMC Molecular Biology
Volume 10

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Guindalini C
Andersen ML
Castro RM
Tufik S

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Tufik S
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Research article

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

Kil S Lee¹ , Tathiana A Alvarenga² , Camila Guindalini² , Monica L Andersen² , Rosa MRPS Castro² and Sergio Tufik²

¹Associação Fundo de Incentivo à Psicofarmacologia (AFIP), Rua Napoleão de Barros, 925, Vila Clementino, São Paulo 04024-002, SP, Brazil

²Department of Psychobiology, Universidade Federal de São Paulo (UNIFESP), Rua Napoleão de Barros, 925, Vila Clementino, São Paulo 04024-002, SP, Brazil

author email corresponding author email

BMC Molecular Biology 2009, 10:45doi:10.1186/1471-2199-10-45


Published:	15 May 2009

Abstract

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.