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

Yasin Panahi,Mandana Hadi Jafari,Zahra Ghasemi,Mohamad Eskandari,Reza Shervin Badv,Mehrdad Pedram,Fahimeh Salasar Moghaddam

doi:10.3390/ijms17101711

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.

Highlights

Autism spectrum disorders (ASD) are serious, lifelong pervasive neurodevelopmental disorders characterized by impairments in reciprocal social interaction, including verbal and non-verbal communication, and repetitive stereotyped behavioral patterns [1]
The present study was prompted by an earlier experiment, during which saliva samples were obtained from five lab members and β-actin gene (ACTB) mRNA levels were measured for RNA quality analysis
It should be noted that ACTB has been used previously by other investigators for RNA quality and transcriptome analysis by Reverse transcriptase quantitative real-time PCR (RT-qPCR) in saliva [35–38], and that it has been used as a common internal control for gene expression studies in ASD (Table 1)

Summary

Introduction

Autism spectrum disorders (ASD) are serious, lifelong pervasive neurodevelopmental disorders characterized by impairments in reciprocal social interaction, including verbal and non-verbal communication, and repetitive stereotyped behavioral patterns [1]. Due to the wide range of genetic heterogeneity reported in ASD, it is quite possible that different causes and various underlying molecular mechanisms may lead to a common set of changes in the brain resulting in a similar behavioral profile [5,6]. Careful comparison of the gene expression profiles in autistic patients with normal subjects could provide important clues for the development of autism and the molecular mechanisms and crossroads involved. An important caveat to consider for selection of reference genes, which are typically selected from housekeeping genes (HKGs), is that expression of HKGs could vary significantly under different biological conditions including disease states, age, sex, drug treatment. Selection of appropriate reference genes for a complex neurodevelopmental disorder such as autism requires very careful design and planning

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Results

Conclusion