Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue

Pors, Susanne Elisabeth; Nikiforov, Dmitry; Zheng, Mengxue; Subiran, Cristina; Bøtkjær, Jane Alrø; Mamsen, Linn Salto; Kristensen, Stine Gry; Andersen, Claus Yding

doi:10.3390/ijms23020886

Cited by 15 publications

(8 citation statements)

References 31 publications

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“…Several previous studies have been using GAPDH as a reference gene. According to Cadenas, J et al ( 2022), GAPDH was found to be the most stable gene for this type of tissue [20]. Also, according to Nikishin et al, they found that levels of GAPDH, ACTB, and HSP90 proteins are similar in native and vitrified/thawed ovarian samples [21].…”

Section: Rna Isolation Cdna Synthesis and Qrt-pcrmentioning

confidence: 97%

Assessment of galectins -1, -3, -4, -8, and -9 expression in ovarian carcinoma patients with clinical implications

et al. 2022

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Background and aim Galectins have been recently tackled by many researchers in the field of cancer due to their role in tumorigenesis, disease progression, and metastasis. Thus, they are currently involved in biomarkers research on several types of cancer. In ovarian cancers, few studies were carried out to evaluate galectins expression profiling. Hence, our present study was executed to evaluate the mRNA expression of galectins -1, -3, -4, -8, and -9 in epithelial ovarian cancers. Methods Fifty-six tumor samples of ovarian carcinomas were analyzed for mRNA expression using qRT-PCR, and fold-changes were calculated in comparison to tissue samples of 26 women with normal ovaries. Results The results of the present paper emphasize the importance of galectins as predictors for targeted therapy. LGALS1, LGALS3, LGALS4, LGALS8, and LGALS9 were found to be mostly overexpressed in ovarian carcinoma patients with the following percentage: 78.6%, 92.9%, 66.1%, 87.5%, and 85.7% respectively. Moreover, galectins -3 and -9 were found to be significantly elevated with lymph node metastasis (p = 0.044 and p = 0.011). Also, upregulation of galectin-1 and -9 were statistically significant in stages IIB, IIC, and IIIB (p = 0.002) in FIGO staging. CA19.9 is positively correlated to galectin-4 expression (p = 0.039). Conclusion Our findings strengthen the role of galectins in carcinogenesis, disease progression, and lymphnode metastasis in ovarian carcinomas. And since these galectins are mostly overexpressed, they could be promising markers for targeted therapy to reduce disease progression and metastasis process.

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Section: Rna Isolation Cdna Synthesis and Qrt-pcrmentioning

confidence: 97%

Assessment of galectins -1, -3, -4, -8, and -9 expression in ovarian carcinoma patients with clinical implications

et al. 2022

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“…Previous studies have reported relatively high variability for the expression of WU-HKGs in a varied range of tissues and organs in livestock animals ( Gromboni et al, 2020 ; Lozano-Villegas et al, 2021 ) as well as in humans ( Mahoney et al, 2004 ; Caracausi et al, 2017 ; Cadenas et al, 2022 ), mice ( Fu et al, 2020 ; Muñoz et al, 2021 ), and insects ( Shi et al, 2016 ), among others.…”

Section: Resultsmentioning

confidence: 99%

“…They suggested the combination of Ywhaz , Hprt1 , and Hmbs as the best set of reference genes for ovarian and uterine tissues of laying hens under control and heat stress conditions. Cadenas et al (2022) found that the stability of all reference genes differs among ovarian cell types in humans. They identified Actb as the best reference gene for oocytes and cumulus cells and B2m for medulla tissue and isolated follicles.…”

Section: Resultsmentioning

confidence: 99%

Investigation of chicken housekeeping genes using next-generation sequencing data

et al. 2022

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Accurate normalization of the gene expression assays, using housekeeping genes (HKGs), is critically necessary. To do so, selection of a proper set of HKGs for a specific experiment is of great importance. Despite many studies, there is no consensus about the suitable set of HKGs for implementing in the quantitative real-time PCR analyses of chicken tissues. A limited number of HKGs have been widely used. However, wide utilization of a little number of HKGs for all tissues is challenging. The emergence of high-throughput gene expression RNA-seq data has enabled the simultaneous comparison of the stability of multiple HKGs. Therefore, employing the average coefficient of variations of at least three datasets per tissue, we sorted all reliably expressed genes (REGs; with FPKM ≥ 1 in at least one sample) and introduced the top 10 most suitable and stable reference genes for each of the 16 chicken tissues. We evaluated the consistency of the results of five tissues using the same methodology on other datasets. Furthermore, we assessed 96 previously widely used HKGs (WU-HKGs) in order to challenge the accuracy of the previous studies. The New Tuxedo software suite was used for the main analyses. The results revealed novel, different sets of reference genes for each of the tissues with 17 common genes among the top 10 genes lists of 16 tissues. The results did disprove the suitability of WU-HKGs such as Actb, Ldha, Scd, B2m, and Hprt1 for any of the tissues examined. On the contrary, a total of 6, 13, 14, 23, and 32 validated housekeeping genes (V-HKGs) were discovered as the most stable and suitable reference genes for muscle, spleen, liver, heart, and kidney tissues, respectively. Although we identified a few new HKGs usable for multiple tissues, the selection of suitable HKGs is required to be tissue specific. The newly introduced reference genes from the present study, despite lacking experimental validation, will be able to contribute to the more accurate normalization for future expression analysis of chicken genes.

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“…Predesigned TaqMan® gene expression assays were performed using following genes: Murine Vascular Endothelial Growth Factor Alpha ( Vegfα), Angiopoietin 1 (Angpt1), Angiopoietin 2 ( Angpt2), Angiopoietin-like 4 (Angptl4) and human B-Cell Lymphoma 2 (BCL2) and BCL-2 associated X protein (BAX), (probe-id: Vegfα, Mm00437306_m1; Angpt1, Mm00456503_m1; Angpt2, Mm00545822_m1; Angptl4, Mm00480431_m1; BCL2 , Hs00608023_m1; BAX , Hs00180269_m1). Glyceraldehyde 3-phosphatdehydrogenase ( Gapdh/GAPDH ) was used as endogenous control for both murine and human gene expression assays [ 44 ] (probe-id: GAPDH, Hs 02786624_g1 ; Gapdh, Mm99999915_m1). The cDNA samples were amplified in duplicates using the LightCycler®480 quantitative PCR instrument (Roche, version 1.5.0.39) as previously described [ 21 , 26 ].…”

Section: Methodsmentioning

confidence: 99%

Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue

Olesen¹,

Pors²,

Adrados³

et al. 2023

Reprod Biol Endocrinol

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Background Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex. Methods Cryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24–36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150–200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks. Results A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045). Conclusions Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.

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Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue

Cited by 15 publications

References 31 publications

Assessment of galectins -1, -3, -4, -8, and -9 expression in ovarian carcinoma patients with clinical implications

Assessment of galectins -1, -3, -4, -8, and -9 expression in ovarian carcinoma patients with clinical implications

Investigation of chicken housekeeping genes using next-generation sequencing data

Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue

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