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Int. J. Mol. Sci. 2016, 17(10), 1711; doi:10.3390/ijms17101711

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

1
Department of Genetics and Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan 45139-56111, Iran
2
Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan 45139-56111, Iran
3
Department of Pediatric Neurology, School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran 14176-13151, Iran
4
Children’s Medical Center, Pediatric Center of Excellence, Tehran University of Medical Sciences (TUMS), Tehran 14176-13151, Iran
5
Metrowest CNS Research Center, Natick, MA 01760, USA
6
Department of Psychiatry, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan 45139-56111, Iran
*
Author to whom correspondence should be addressed.
Academic Editor: Stephen A. Bustin
Received: 9 August 2016 / Revised: 29 September 2016 / Accepted: 30 September 2016 / Published: 12 October 2016
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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 naïve 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. View Full-Text
Keywords: childhood autism; non-syndromic; transcriptome; saliva; reverse transcriptase quantitative real-time PCR (RT-qPCR); housekeeping genes (HKGs); reference gene; stability of expression; geNorm; NormFinder childhood autism; non-syndromic; transcriptome; saliva; reverse transcriptase quantitative real-time PCR (RT-qPCR); housekeeping genes (HKGs); reference gene; stability of expression; geNorm; NormFinder
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MDPI and ACS Style

Panahi, Y.; Salasar Moghaddam, F.; Ghasemi, Z.; Hadi Jafari, M.; Shervin Badv, R.; Eskandari, M.R.; Pedram, M. Selection of Suitable Reference Genes for Analysis of Salivary Transcriptome in Non-Syndromic Autistic Male Children. Int. J. Mol. Sci. 2016, 17, 1711.

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