title: Validation of commonly used reference genes for sleep-related gene expression studies.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Associação Fundo de Incentivo à Psicofarmacologia, São Paulo, Brazil. kil_sun_lee@yahoo.com.br
authors.forename: Kil S
authors.initials: KS
authors.lastname: Lee
authors.forename: Tathiana A
authors.initials: TA
authors.lastname: Alvarenga
authors.forename: Camila
authors.initials: C
authors.lastname: Guindalini
authors.forename: Monica L
authors.initials: ML
authors.lastname: Andersen
authors.forename: Rosa M R P S
authors.initials: RM
authors.lastname: Castro
authors.forename: Sergio
authors.initials: S
authors.lastname: Tufik
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D001921:Brain / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012892:Sleep Deprivation / Q000235:genetics* / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000235:genetics*
chemical_list: D000199:Actins; D019208:Brain-Derived Neurotrophic Factor; D001613:beta 2-Microglobulin; D005993:Glycerolphosphate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase