TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells

Jang, Sang Ock; Jeon, Ryoung-Hoon; Kim, Hwan-Deuk; Hwang, Jong-Chan; Lee, Hyeon-Jeong; Bae, Seulgi; Lee, Sung‐Lim; Rho, Gyu‐Jin; Kim, Seung-Joon; Lee, Won–Jae

doi:10.5713/ajas.20.0238

Cited by 15 publications

(19 citation statements)

References 30 publications

(42 reference statements)

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“…It has been shown that RPS15A and RPS9 were the most stable internal reference genes in the Indian buffalo and bovine peripheral blood mononuclear cells 7 . RPL15 was also the most stable internal reference in bovine oocytes collected in winter and summer 28 , while RPL4 was the most stable internal reference gene in bovine bone marrow mesenchymal stem cell differentiation 32 . RPS9 and RPL19 in bovine maternal reproductive tissues and fetal tissues were found to be stably expressed 33 .…”

Section: Discussionmentioning

confidence: 99%

Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells

Wang

Liang

et al. 2022

Sci Rep

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The accuracy of sixteen commonly used internal reference genes was assessed in skeletal muscle-derived satellite cells of Qinchuan cattle at different stages of proliferation and induction of differentiation to determine the most suitable ones. Quantitative real-time PCR and three commonly used algorithmic programs, GeNorm, NormFinder and BestKeeper, were used to evaluate the stability of expression of the candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K , RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) in skeletal muscle-derived satellite cells at 0, 12, 24, 36 and 48 h of growth and after differentiation for 0, 2, 4, 6 and 8 days. The expression of two satellite cell marker genes, CCNA2 and MYF5, was used for validation analysis. The results of the software analyses showed that GAPDH and RPS15A were the most stable reference gene combinations during in vitro proliferation of bovine skeletal muscle-derived satellite cells, RPS15A and RPS9 were the most stable reference gene combinations during in vitro induction of differentiation of the cells, and PPIA was the least stable reference gene during proliferation and differentiation and was not recommended. This study lays the foundation for the selection of reference genes for qRT-PCR during the proliferation and induction of differentiation of bovine skeletal muscle-derived satellite cells.

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Section: Discussionmentioning

confidence: 99%

Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells

Wang

Liang

et al. 2022

Sci Rep

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“…It suggested that the discrepancy in the ranking of the reference gene was caused by differences in the experimental condition. Hence, before analyzing gene expression by qRT-PCR, selection of the most stable reference gene under particular experimental conditions is an essential step [ 22 , 25 ].…”

Section: Discussionmentioning

confidence: 99%

“…The Bestkeeper index was compared in a pair-wise fashion via Pearson correlation coefficients for evaluating the reference gene stability ranking [ 24 ]. Previously, we analyzed the most stable reference gene in other species using three algorithms [ 21 , 22 , 25 ]. Eight candidate reference genes were estimated by independent statistical algorithms, different stability ranking was obtained.…”

Section: Introductionmentioning

confidence: 99%

HPRT1 Most Suitable Reference Gene for Accurate Normalization of mRNA Expression in Canine Dermal Tissues with Radiation Therapy

Lee

Choe

Han

et al. 2022

Genes

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Reference genes are crucial in molecular biological studies as an internal control for gene re-search as they exhibit consistent expression patterns across many tissue types. In canines, radiation therapy is the most important therapeutic tool to cure various diseases like cancer. However, when using radiation for therapeutic strategy, radiation exposure to healthy tissues leads to some possible side effects such as acute radiation-induced skin injury and alters gene expression. Therefore, the analysis of a change in reference gene expression during the skin recovery process after radiation therapy is essential in healthy canine tissue. In the present study, we analyzed eight reference genes (ACTB, GAPDH, YWHAZ, GUSB, HPRT1, RPL4, RPS5, and TBP) in canine dermal tissues at 0, 1, 2, 3, 4, 5, 7, and 9 weeks of radiation exposure that affected the skin condition of canines. The stability of reference genes is determined by evaluating radiation therapy’s effect on healthy canine dermal tissue. Epidermal marker, Keratin 10 expression varies each week after irradiation, and HPRT1 is found to be the most suitable for normalization of mRNA expression in radiation-exposed canine dermal tissues. Changes in the gene expression level were evaluated by using a reliable tool such as quantitative real-time polymerase chain reaction (qRT-PCR). In order to achieve a valid qRT-PCR result, the most stable reference genes used for normalization after the radiation exposure process are important. Therefore, the current study was designed to evaluate the most stable reference gene for the post-irradiation canine tissues. After radiation exposure, the alternation of reference gene expression was estimated by three algorithms (geNorm, Normfinder, and Bestkeeper). The RG validation programs (GeNorm and NormFinder) suggested that HPRT1, RPL4, and TBP were suitable for normalization in qRT-PCR. Furthermore, three algorithms suggested that HPRT1 was the most stable reference gene for normalization with qRT-PCR results, regardless of before and after radiation exposure. Whereas GAPDH was found to be the most unstable reference gene. In addition, the use of stable or unstable reference genes for the normalization of Keratin 10 expression showed statistical differences. Therefore, we observed that, to obtain accurate and suitable PCR results of the canine tissues with and without radiation exposure, the HPRT1 reference gene is recommended for normalization with its high stability. Additionally, the use of RGs such as HPRT1, RPL4, and TBP for normalization in qRT-PCR experiments is recommended for post-radiation canine tissues to generate more accurate and reliable data. These results will provide fundamental information regarding internal controls for gene expression studies and can be used for the analysis of gene patterns in regenerative medicine.

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“…Specific primers for the selected genes ( Table 2 ) were designed de novo through the Primer-BLAST and AutoDimer online applications [ 29 , 30 ]. TATA-box binding protein (TBP) gene, which is constitutively expressed, was used as a control to normalize the results [ 31 ]. The gene expression units are expressed as relative quantities of mRNA.…”

Section: Methodsmentioning

confidence: 99%

Irregular Expression of Cellular Stress Response Markers in the Placenta of Women with Chronic Venous Disease

et al. 2022

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Pregnancy comprises a period in a woman’s life in which the circulatory system is subjected to hemodynamical and biochemical changes. During this period, while restructuring blood vessels and exchanging maternal-fetal products there is an increased risk of developing chronic venous disease (CVD), which may have an echo in life after childbirth for both mother and child. Previously, we investigated that pregnancy-associated CVD involves changes in placental architecture at angiogenesis, lymphangiogenesis and villi morphology compared with healthy controls (HC) with no history of CVD. We aimed to more deeply investigate the oxidative stress response in placenta from women with CVD versus HC through several markers (NRF2, KEAP1, CUL3, GSK-3β). An observational, analytical, and prospective cohort study was conducted on 114 women in their third trimester of pregnancy (32 weeks). A total of 62 participants were clinically diagnosed with CVD. In parallel, 52 controls with no history of CVD (HC) were studied. Gene and protein expressions of NRF2, KEAP1, CUL3, GSK-3β were analyzed by real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry. Nrf2 gene and protein expression was significantly greater in placental villi of women with CVD, while Keap1, CUL-3 and GSK-3β gene and protein expressions were significantly lower. Our results defined an aberrant gene and protein expression of Nrf2 and some of their main regulators Keap1, CUL-3 and GSK-3 β in the placenta of women with CVD, which could be an indicator of an oxidative environment observed in this tissue.

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TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells

Cited by 15 publications

References 30 publications

Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells

Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells

HPRT1 Most Suitable Reference Gene for Accurate Normalization of mRNA Expression in Canine Dermal Tissues with Radiation Therapy

Irregular Expression of Cellular Stress Response Markers in the Placenta of Women with Chronic Venous Disease

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