Identification of reliable reference genes for quantitative real-time PCR in ovary and uterus of laying hens under heat stress

Hassanpour, Hossein; Aghajani, Zahra; Bahadoran, Shahab; Farhadi, Navid; Nazari, Hassan; Kaewduangta, Waranyoo

doi:10.1080/10253890.2019.1574294

Cited by 19 publications

(27 citation statements)

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“…cDNA was amplified using the Power SYBR Green PCR Master Mix (catalog #4367659, Thermo Fisher Scientific, Waltham, MA, USA). Target gene expression was normalized to hypoxanthine phosphoribosyltransferase 1 (Hprt1, Hassanpour et al, 2019). cDNA from the brain tissue of post-hatched 1-day-old chickens was used to generate standard curves for each gene.…”

Section: Quantitative Real-time Polymerase Chain Reactionmentioning

confidence: 99%

Initiation of Supporting Cell Activation for Hair Cell Regeneration in the Avian Auditory Epithelium: An Explant Culture Model

Matsunaga

Kita

Yamamoto

et al. 2020

Front. Cell. Neurosci.

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Sensorineural hearing loss is a common disability often caused by the loss of sensory hair cells in the cochlea. Hair cell (HCs) regeneration has long been the main target for the development of novel therapeutics for sensorineural hearing loss. In the mammalian cochlea, hair cell regeneration is limited, but the auditory epithelia of non-mammalian organisms retain the capacity for hair cell regeneration. In the avian basilar papilla (BP), supporting cells (SCs), which give rise to regenerated hair cells, are usually quiescent. Hair cell loss induces both direct transdifferentiation and mitotic division of supporting cells. Here, we established an explant culture model for hair cell regeneration in chick basilar papillae and validated it for investigating the initial phase of hair cell regeneration. The histological assessment demonstrated hair cell regeneration via direct transdifferentiation of supporting cells. Labeling with 5-ethynyl-2′-deoxyuridine (EdU) revealed the occurrence of mitotic division in the supporting cells at specific locations in the basilar papillae, while no EdU labeling was observed in newly generated hair cells. RNA sequencing indicated alterations in known signaling pathways associated with hair cell regeneration, consistent with previous findings. Also, unbiased analyses of RNA sequencing data revealed novel genes and signaling pathways that may be related to the induction of supporting cell activation in the chick basilar papillae. These results indicate the advantages of our explant culture model of the chick basilar papillae for exploring the molecular mechanisms of hair cell regeneration.

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Section: Quantitative Real-time Polymerase Chain Reactionmentioning

confidence: 99%

Initiation of Supporting Cell Activation for Hair Cell Regeneration in the Avian Auditory Epithelium: An Explant Culture Model

Matsunaga

Kita

Yamamoto

et al. 2020

Front. Cell. Neurosci.

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“…The higher the M value is, the worse the stability of gene expression; in contrast, the lower the M value is, the better the stability of gene expression (Vandesompele et al, 2002). To date, some reference genes have been validated for the standardization of RT-qPCR data in chickens, but these genes are only concentrated in the gene expression analysis of a certain tissue or cell, such as muscle, brain, abdominal fat, heart, lung, ovary, uterus, lymphoid organ, articular cartilage, chicken embryo fibroblasts, IEL-NK cell, and DT40 cell line (Yang et al, 2013;Bagés et al, 2015;Nascimento et al, 2015;Mitra et al, 2016;Staines et al, 2016;Katarzyńska-Banasik et al, 2017;Hassanpour et al, 2018Hassanpour et al, , 2019Simon et al, 2018;Boo et al, 2020;Dunislawska et al, 2020;Hul et al, 2020;Marciano et al, 2020). Whether these reference genes are also suitable to the study of gene expression during the growth and development of a specific tissue or cell in chickens, such as adipose tissue and adipocyte, still needs to be further determined.…”

Section: Discussionmentioning

confidence: 99%

“…Simon et al (2018) analyzed the expression stability of 10 reference genes in the hypothalamus of chickens under three different nutritional conditions using BestKeeper, GeNorm, NormFinder, Ct, and a multivariate linear mixed-effect modeling algorithm, and found that TBP was one of the most stable reference gene in the hypothalamus of chickens. Bagés et al (2015) and Hassanpour et al (2019) also identified TBP as an stably expressed reference gene in chicken muscle, liver, ovary, and uterus tissue using GeNorm, NormFinder, CV, and BestKeeper methods. HMBS, the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB; Bung et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…HMBS, the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB; Bung et al, 2018). HMBS has been identified as a stably expressing reference gene in many tissues of chickens and is also a commonly used reference gene in chicken RT-qPCR experiments (Cedraz de Oliveira et al, 2017;Lenart et al, 2017;Hassanpour et al, 2019;Boo et al, 2020). Note that only male birds were used in the present study for the selection procedure requirement of the NEAUHLF (Guo et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

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Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes

et al. 2021

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Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens.

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“…In chickens, studies evaluating gene stability in several tissues, such as muscle, embryo, ovary, uterus, lung and heart fibroblasts have been reported [ 35 , 37 , 42 – 45 ]. However, there are no studies regarding the stability of endogenous genes in poultry articular cartilage tissue.…”

Section: Discussionmentioning

confidence: 99%

Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

et al. 2020

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The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies.

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Identification of reliable reference genes for quantitative real-time PCR in ovary and uterus of laying hens under heat stress

Cited by 19 publications

References 50 publications

Initiation of Supporting Cell Activation for Hair Cell Regeneration in the Avian Auditory Epithelium: An Explant Culture Model

Initiation of Supporting Cell Activation for Hair Cell Regeneration in the Avian Auditory Epithelium: An Explant Culture Model

Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes

Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

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