Reference genes for reverse transcription quantitative PCR in canine brain tissue

Stassen, Quirine E. M.; Riemers, Frank M.; Reijmerink, Hannah; Leegwater, P.A.J.; Penning, Louis C.

doi:10.1186/s13104-015-1628-4

Cited by 21 publications

(23 citation statements)

References 35 publications

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“…For data normalization, the mRNA expression levels of five reference genes were analysed: signal recognition particle receptor, succinate dehydrogenase complex subunit A, ribosomal protein S5, hypoxanthine‐guanine phosphoribosyltransferase and tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein zeta …”

Section: Methodscontrasting

confidence: 68%

Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Sanders

Staalduinen

Uijens

et al. 2019

Vet Comparative Oncology

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Hypercortisolism is caused by a cortisol-secreting adrenocortical tumour (ACT) in approximately 15%-20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol-secreting ACTs were included from 40 dogs, of which follow-up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate-high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT-PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour-transforming gene-1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor-1 (SF-1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets. K E Y W O R D S adrenocortical adenoma, adrenocortical carcinoma, cancer, canine, Cushing syndrome, prognostic, treatment targets

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Section: Methodscontrasting

confidence: 68%

Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Sanders

Staalduinen

Uijens

et al. 2019

Vet Comparative Oncology

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“…The qPCR reactions contained 1x IQ SYBRGreen SuperMix (Bio-Rad, Laboratories, Hercules, CA), 400 nM of each primer and 1 μl cDNA, obtained as described above, in a total volume of 15 μl. The reference genes were RPS19 and RPL13A , which were analyzed as described [29]. The reactions were performed in a MyiQ2 thermal cycler and the data was analyzed with IQ5 software (both from Bio-Rad, Laboratories, Hercules, CA).…”

Section: Methodsmentioning

confidence: 99%

Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7

et al. 2016

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BackgroundInbreeding and population bottlenecks in the ancestry of Friesian horses has led to health issues such as dwarfism. The limbs of dwarfs are short and the ribs are protruding inwards at the costochondral junction, while the head and back appear normal. A striking feature of the condition is the flexor tendon laxity that leads to hyperextension of the fetlock joints. The growth plates of dwarfs display disorganized and thickened chondrocyte columns. The aim of this study was to identify the gene defect that causes the recessively inherited trait in Friesian horses to understand the disease process at the molecular level.ResultsWe have localized the genetic cause of the dwarfism phenotype by a genome wide approach to a 3 Mb region on the p-arm of equine chromosome 14. The DNA of two dwarfs and one control Friesian horse was sequenced completely and we identified the missense mutation ECA14:g.4535550C > T that cosegregated with the phenotype in all Friesians analyzed. The mutation leads to the amino acid substitution p.(Arg17Lys) of xylosylprotein beta 1,4-galactosyltransferase 7 encoded by B4GALT7. The protein is one of the enzymes that synthesize the tetrasaccharide linker between protein and glycosaminoglycan moieties of proteoglycans of the extracellular matrix. The mutation not only affects a conserved arginine codon but also the last nucleotide of the first exon of the gene and we show that it impedes splicing of the primary transcript in cultured fibroblasts from a heterozygous horse. As a result, the level of B4GALT7 mRNA in fibroblasts from a dwarf is only 2 % compared to normal levels. Mutations in B4GALT7 in humans are associated with Ehlers-Danlos syndrome progeroid type 1 and Larsen of Reunion Island syndrome. Growth retardation and ligamentous laxity are common manifestations of these syndromes.ConclusionsWe suggest that the identified mutation of equine B4GALT7 leads to the typical dwarfism phenotype in Friesian horses due to deficient splicing of transcripts of the gene. The mutated gene implicates the extracellular matrix in the regular organization of chrondrocyte columns of the growth plate. Conservation of individual amino acids may not be necessary at the protein level but instead may reflect underlying conservation of nucleotide sequence that are required for efficient splicing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3186-0) contains supplementary material, which is available to authorized users.

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“…Quantitative Real-Time PCR reactions were performed on a StepOne Plus Instrument (Thermo Fisher Scientific) using commercial TaqMan assays and the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). The following TaqMan assays were used to target three genes, which were previously validated as reference genes for dog brain samples [23]: Cf04419463_gH for GAPDH;…”

Section: °Cmentioning

confidence: 99%

“…As the molecular quality of stored tissue samples is a major question, we first assessed this by measuring the gene expression stability of house keeping genes in brain and muscle collected under our protocol. We chose three endogenous reference genes that were previously validated in dog brain samples (GAPDH, HPRT1 and HMBS, [23]). Our hypothesis was that the variance in the detected expression stability values of these genes, determined by BestKeeper and NormFinder, would be similar to the results shown by previous studies [23,24], if the obtainment and purification procedures applied in the CBTB were efficient and met the quality standards.…”

mentioning

confidence: 99%

“…We chose three endogenous reference genes that were previously validated in dog brain samples (GAPDH, HPRT1 and HMBS, [23]). Our hypothesis was that the variance in the detected expression stability values of these genes, determined by BestKeeper and NormFinder, would be similar to the results shown by previous studies [23,24], if the obtainment and purification procedures applied in the CBTB were efficient and met the quality standards. By investigating muscle samples in addition to brain tissue, we expected to find similar, or at least not greater, BestKeeper SD values, as reported previously for whole body tissues [24].…”

mentioning

confidence: 99%

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Establishing a canine brain and tissue bank – molecular validation by RT-qPCR targeting three reference genes

Sándor

Tátrai

Czeibert

et al. 2019

Preprint

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Background: Dogs ( Canis familiaris ) are natural models of several human diseases, including age-related dementia. However, the molecular techniques, which are routinely applied in invertebrate and rodent models to study disease pathologies and mechanisms, has limited applicability in dogs, mainly because of ethical reasons. In the case of humans, the limited accessibility of tissue samples is at least partly solved by biobanks, which collect and store tissues and organs from voluntary donations. A similar approach with pet dogs could support both translational and veterinary research goals by providing access to good quality biological materials obtained from a wide range of dogs with known ancestry, life history and medical background. Therefore, we have established an initiative, the Canine Brain and Tissue Bank, to collect and store biological samples from pet dogs. Objectives: The molecular qualities of tissue samples collected and stored on a tissue bank are crucial for reliable downstream applications. We used quantitative Real-Time PCR methodology to assess the stability of mRNA content in our stored samples. Three previously validated reference genes, GAPDH , HMBS and HPRT1 were chosen as targets. Results: The tested reference genes showed expression patterns and stability values that were consistent with the literature. Conclusions: Based on the results, the molecular quality of tissues collected in the CBTB’s donations system can fit in the standards of dog gene expression analyses.

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Reference genes for reverse transcription quantitative PCR in canine brain tissue

Cited by 21 publications

References 35 publications

Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7

Establishing a canine brain and tissue bank – molecular validation by RT-qPCR targeting three reference genes

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