Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

Hul, Ludmila Mudri; Ibelli, A. M. G.; Peixoto, J. de O.; Souza, Mayla Regina; Savoldi, Igor Ricardo; Marcelino, Débora Ester Petry; Tremea, Mateus; Ledur, Mônica Corrêa

doi:10.1371/journal.pone.0238189

Cited by 8 publications

(5 citation statements)

References 80 publications

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“…The stability rank order in the tissue groups was different from that observed in the acaricide treatment groups, with α‐tubulin and RpL5 being the most stable and Actin and eEF2 being the least stable. α‐tubulin encodes a major component of the cytoskeleton and has been widely accepted as a reference gene across various species (P. Liang et al, 2014; Rentoft et al, 2010; Sun et al, 2010) along with RpL5 , which encodes a ribosomal subunit protein (Gromboni et al, 2020; Hul et al, 2020). Both α‐tubulin and RpL5 showed good performance in all the cross‐tissue comparisons, except for the cuticle‐gut/fat body comparison, indicating that the application of both genes as reference genes for cross‐tissue comparison is reliable.…”

Section: Discussionmentioning

confidence: 99%

Selection of stable reference genes for quantitative real‐time PCR in the Varroa mite, Varroa destructor

Lee

Kim

et al. 2022

Arch Insect Biochem Physiol

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To investigate the acaricide toxicity and resistance mechanisms in the Varroa mite, it is essential to understand the genetic responses of Varroa mites to acaricides, which are usually evaluated by transcriptional profiling based on quantitative real‐time polymerase chain reaction (qPCR). In this study, to select reference genes showing consistent expression patterns regardless of the acaricide treatment or the type of tissue, Varroa mites treated with each of the three representative acaricides (coumaphos, fluvalinate, and amitraz) were processed for transcriptomic analysis, from which eight genes (NADH dehydrogenase [NADHD], glyceraldehyde‐3‐phosphate dehydrogenase [GAPDH], eukaryotic translation elongation factor 1 α 1 [eEF1A1], eukaryotic translation elongation factor 2 [eEF2], ribosomal protein L5 [RpL5], Actin, tubulin α‐1D chain [α‐tubulin], and Rab1) were selected as candidates. The transcription profiles of these genes, depending on the treatment of the three acaricides or across different tissues (cuticle, legs, gut/fat bodies, and synganglion), were analyzed using qPCR with four validation programs, BestKeeper, geNorm, NormFinder, and RefFinder. Following acaricide treatment, eEF1A1 and NADHD showed the least variation in their expression levels, whereas the expression levels of α‐tubulin and RpL5 were the most stable across different tissue groups. Rab1/GAPDH and Actin/eEF2 showed the least stable expression patterns following acaricide treatments and across different tissues, respectively, requiring precautions for use. When vitellogenin gene expression was analyzed by different reference genes, its expression profiles varied significantly depending on the reference genes, highlighting the importance of proper reference gene use. Thus, it is recommended using eEF1A1 and NADHD as reference genes for the comparison of the effects of acaricide on the whole body, whereas α‐tubulin and RpL5 are recommended for investigating the tissue‐specific expression profiles of target genes.

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Section: Discussionmentioning

confidence: 99%

Selection of stable reference genes for quantitative real‐time PCR in the Varroa mite, Varroa destructor

Lee

Kim

et al. 2022

Arch Insect Biochem Physiol

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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%

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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“…Al-Sabah also pointed out that RPL4 is also one of the most stable reference genes in articular cartilage under mechanical stress (Al-Sabah et al, 2016). At the same time, there are still a large number of studies showing that RPL5 is the most stable reference gene in various tissues, such as murine cornea (Ren et al, 2010), right heart failure tissue from human (Li et al, 2017), breast muscle of chicken (Marciano et al, 2020) and broilers cartilage (Hul et al, 2020). The above results are consistent with our results, indicating that RPL4 and RPL5 have high stability for RT-qPCR assay in a variety of tissues, including cartilage in the development.…”

Section: Discussionmentioning

confidence: 99%

Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage

Han

et al. 2020

Front. Genet.

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Real-time fluorescence quantitative PCR (RT-qPCR) is widely used to detect gene expression levels, and selection of reference genes is crucial to the accuracy of RT-qPCR results. Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that using the panel of reference genes for RT-qPCR is conducive to obtaining accurate experimental results. However, the selection of the panel of reference genes for RT-qPCR in rat developing cartilage has not been well documented. In this study, we selected eight reference genes commonly used in rat cartilage from literature (GAPDH, ACTB, 18S, GUSB, HPRT1, RPL4, RPL5, and SDHA) as candidates. Then, we screened out the optimal panel of reference genes in female and male rat cartilage of fetus (GD20), juvenile (PW6), and puberty (PW12) in physiology with stability analysis software of genes expression. Finally, we verified the reliability of the selected panel of reference genes with the rat model of intrauterine growth retardation (IUGR) induced by prenatal dexamethasone exposure (PDE). The results showed that the optimal panel of reference genes in cartilage at GD20, PW6, and PW12 in physiology was RPL4 + RPL5, which was consistent with the IUGR model, and there was no significant gender difference. Further, the results of standardizing the target genes showed that RPL4 + RPL5 performed smaller intragroup differences than other panels of reference genes or single reference genes. In conclusion, we found that the optimal panel of reference genes in female and male rat developing cartilage was RPL4 + RPL5, and there was no noticeable difference before and after birth.

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Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

Cited by 8 publications

References 80 publications

Selection of stable reference genes for quantitative real‐time PCR in the Varroa mite, Varroa destructor

Selection of stable reference genes for quantitative real‐time PCR in the Varroa mite, Varroa destructor

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

Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage

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