Validation of adequate endogenous reference genes for reverse transcription-qPCR studies in human post-mortem brain tissue of SIDS cases

Abstract

Sudden infant death syndrome (SIDS) is the main cause of post-neonatal infant death in most developed countries. It is still of ambiguous etiology. Gene expression studies of relevant target genes using reverse transcription quantitative real-time PCR (RT-qPCR) in SIDS cases, and comparing them with age-matched controls, could help in understanding the pathogenesis of SIDS. However, selecting inadequate reference genes used for normalization of the RT-qPCR gene expression data can give misleading results. The aim of the present study was to identify reference genes with the most stable expression in post-mortem brainstem samples of SIDS and control cases. Among the five candidate reference genes (GAPDH, GUSB, HMBS, SDHA, UBXN6) studied in both groups, SDHA and UBXN6 were identified as the most stable. To further demonstrate the importance of using validated genes for RT-qPCR data normalization, the expression of a potential gene of interest in SIDS, the RPS27A gene, was evaluated using validated versus non-validated reference genes for normalization. This gene encodes the ubiquitin protein that has been shown in other pathological studies to be induced in SIDS. Using the identified most stable genes for normalization of RPS27A gene expression data revealed, as expected, a statistically significant up-regulation in SIDS as compared to the controls. However, using a single unstable reference gene for normalization resulted in no significant differences in transcript abundance of RPS27A between SIDS and the controls. This emphasizes the need for validation of the suitability of reference genes used in a given tissue type under certain experimental conditions.

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

“…This assumption was challenged by observing that a subgroup of mammalian mRNA transcripts with the AUUUA motif in the 3′ untranslated region are particularly susceptible to PMI-related degradation [ 32 ]. Since then, a growing body of evidence has indicated that selective gene expression or uneven half-lives of transcripts are characteristic of postmortem human tissues [ 29 , 33 , 34 ]. Part of this variation is attributable to hypoxic stresses occurring after somatic death that result in altered gene expression profiles, including up-regulation of certain genes (hypoxic inducible factor—HIF [ 23 , 35 ], cytoskeleton-related genes [ 29 ], Hsp70 [ 36 , 37 ], Bag1 [ 38 ], Gapdh [ 31 ]) and down-regulation of serine protease inhibitors [ 29 , 39 ], Nos3 and related genes [ 40 ].…”

Reduced collagen accumulation and augmented MMP-2 activity in left ventricle of old rats submitted to high-intensity resistance training

“…The target gene expression was normalized to ribosomal protein S27a (RPS27A) mRNA. This gene encodes a fusion protein consisting of ubiquitin and ribosomal protein S27a (20).…”

No association to sudden infant death syndrome detected by targeted amplicon sequencing of 24 genes

“…Various mutations and polymorphisms in several genes have been found associated with SIDS, but so far only mutations in genes involved in cardiac arrhythmias and fatty acid oxidation defects can be considered independent cause of death. 6,7 A few studies have investigated messenger ribonucleic acid (mRNA) expression in SIDS, and mRNA status for different genes has been reported.. [8][9][10] This information has provided important insight to unfavourable genetic regulation prior to death. Also, the information has helped localise candidate genes, in which specific gene variants and polymorphisms might be regarded as risk factors for SIDS.…”

“…A few studies have investigated messenger ribonucleic acid (mRNA) expression in SIDS, and mRNA status for different genes has been reported. 8‐10 . This information has provided important insight to unfavourable genetic regulation prior to death.…”