Validation of Rat Reference Genes for Improved Quantitative Gene Expression Analysis Using Low Density Arrays

Cai, Jenny Hong; Deng, Shibing; Kumpf, Steven W.; Lee, Patricia A.; Zagouras, Panayiotis; Ryan, Anne; Gallagher, D. S.

doi:10.2144/000112400

Cited by 29 publications

(16 citation statements)

References 20 publications

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“…We found that PPIB showed less variation than beta-actin and GAPDH in rat spleen tissue (Figure S2). PPIB as a reliable reference gene in rat spleen mRNA analysis was compatible with another previous report [17]. Thereafter, we used PPIB as a reference gene in subsequent studies.…”

Section: Resultssupporting

confidence: 66%

Prenatal Dexamethasone and Postnatal High-Fat Diet Decrease Interferon Gamma Production through an Age-Dependent Histone Modification in Male Sprague-Dawley Rats

Tain

Sheen

et al. 2016

IJMS

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Overexposure to prenatal glucocorticoid (GC) disturbs hypothalamic-pituitary-adrenocortical axis-associated neuroendocrine metabolism and susceptibility to metabolic syndrome. A high-fat (HF) diet is a major environmental factor that can cause metabolic syndrome. We aimed to investigate whether prenatal GC plus a postnatal HF diet could alter immune programming in rat offspring. Pregnant Sprague-Dawley rats were given intraperitoneal injections of dexamethasone or saline at 14–21 days of gestation. Male offspring were then divided into four groups: vehicle, prenatal dexamethasone exposure, postnatal HF diet (VHF), and prenatal dexamethasone exposure plus a postnatal HF diet (DHF). The rats were sacrificed and adaptive immune function was evaluated. Compared to the vehicle, the DHF group had lower interferon gamma (IFN-γ) production by splenocytes at postnatal day 120. Decreases in H3K9 acetylation and H3K36me3 levels at the IFN-γ promoter correlated with decreased IFN-γ production. The impaired IFN-γ production and aberrant site-specific histone modification at the IFN-γ promoter by prenatal dexamethasone treatment plus a postnatal HF diet resulted in resilience at postnatal day 180. Prenatal dexamethasone and a postnatal HF diet decreased IFN-γ production through a site-specific and an age-dependent histone modification. These findings suggest a mechanism by which prenatal exposure to GC and a postnatal environment exert effects on fetal immunity programming.

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Section: Resultssupporting

confidence: 66%

Prenatal Dexamethasone and Postnatal High-Fat Diet Decrease Interferon Gamma Production through an Age-Dependent Histone Modification in Male Sprague-Dawley Rats

Tain

Sheen

et al. 2016

IJMS

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“…Single reference gene normalization across developmental stages may be difficult because of the wide variation in expression at different developmental stages [14]. We show here a marked shift in the expression stability of several reference genes in the transition from neonatal to adult rat myocardium.…”

Section: Discussionmentioning

confidence: 72%

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

et al. 2010

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BackgroundQuantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.ResultsThe abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions.ConclusionsThe most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

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“…The set of reference genes examined in our study were chosen, because of their previous use to normalization changes in gene expression. For example, MAPK6 and GAPD have been found to be stable normalization genes in rat brain tissue (Cai et al 2007). Use of 18S RNA and actin have been highly published in the literature.…”

Section: Discussionmentioning

confidence: 99%

Quantification of Gene Expression after Painful Nerve Injury: Validation of Optimal Reference Genes

et al. 2011

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Stably expressed housekeeping genes (HKGs) are necessary for standardization of transcript measurement by quantitative real time PCR (qRT-PCR). Peripheral nerve injury disrupts expression of numerous genes in sensory neurons, but the stability of conventional HKGs has not been tested in this context. We examined the stability of candidate HKGs during nerve injury, including the commonly used 18s ribosomal RNA (18s rRNA), β tubulin I (Tubb5) and β tubulin III (Tubb3), actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase 1 (HPRT1), and mitogen activated protein kinase 6 (MAPK6). Total RNA for cDNA synthesis was isolated from dorsal root ganglia of rats at 3, 7 and 21 days following either skin incision alone or spinal nerve ligation, after which the axotomized and adjacent ganglia were analyzed separately. Relative stability of HKGs was determined using statistical algorithms geNorm and NormFinder. Both analyses identified MAPK6 and GAPDH as the two most stable HKGs for normalizing gene expression for qRT-PCR analysis in the context of peripheral nerve injury. Our findings indicate that a priori analysis of HKG expression levels is important for accurate normalization of gene expression in models of nerve injury.

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Validation of Rat Reference Genes for Improved Quantitative Gene Expression Analysis Using Low Density Arrays

Cited by 29 publications

References 20 publications

Prenatal Dexamethasone and Postnatal High-Fat Diet Decrease Interferon Gamma Production through an Age-Dependent Histone Modification in Male Sprague-Dawley Rats

Prenatal Dexamethasone and Postnatal High-Fat Diet Decrease Interferon Gamma Production through an Age-Dependent Histone Modification in Male Sprague-Dawley Rats

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

Quantification of Gene Expression after Painful Nerve Injury: Validation of Optimal Reference Genes

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