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Selection of Suitable Reference Genes for Analysis of Salivary Transcriptome in Non-Syndromic Autistic Male Children Publisher Pubmed



Panahi Y1 ; Moghaddam FS2 ; Ghasemi Z2 ; Jafari MH1 ; Badv RS3, 4 ; Eskandari MR5, 6 ; Pedram M1
Authors
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Authors Affiliations
  1. 1. Department of Genetics and Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, 45139-56111, Iran
  2. 2. Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, 45139-56111, Iran
  3. 3. Department of Pediatric Neurology, School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, 14176-13151, Iran
  4. 4. Children’s Medical Center, Pediatric Center of Excellence, Tehran University of Medical Sciences (TUMS), Tehran, 14176-13151, Iran
  5. 5. Metrowest CNS Research Center, Natick, 01760, MA, United States
  6. 6. Department of Psychiatry, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, 45139-56111, Iran

Source: International Journal of Molecular Sciences Published:2016


Abstract

Childhood autism is a severe form of complex genetically heterogeneous and behaviorally defined set of neurodevelopmental diseases, collectively termed as autism spectrum disorders (ASD). Reverse transcriptase quantitative real-time PCR (RT-qPCR) is a highly sensitive technique for transcriptome analysis, and it has been frequently used in ASD gene expression studies. However, normalization to stably expressed reference gene(s) is necessary to validate any alteration reported at the mRNA level for target genes. The main goal of the present study was to find the most stable reference genes in the salivary transcriptome for RT-qPCR analysis in non-syndromic male childhood autism. Saliva samples were obtained from nine drug naive non-syndromic male children with autism and also sex-, age-, and location-matched healthy controls using the RNA-stabilizer kit from DNA Genotek. A systematic two-phased measurement of whole saliva mRNA levels for eight common housekeeping genes (HKGs) was carried out by RT-qPCR, and the stability of expression for each candidate gene was analyzed using two specialized algorithms, geNorm and NormFinder, in parallel. Our analysis shows that while the frequently used HKG ACTB is not a suitable reference gene, the combination of GAPDH and YWHAZ could be recommended for normalization of RT-qPCR analysis of salivary transcriptome in non-syndromic autistic male children. © 2016 by the authors; licensee MDPI, Basel, Switzerland.