Analysis of stability of reference genes for qPCR in bovine preadipocytes during proliferation and differentiation in vitro

Wang, Guohua; Wang, Si-Hu; Zhang, Wenzhen; Liang, Chengcheng; Chen, Gong; Wang, Xiaoyu; Liu, Zan

doi:10.1016/j.gene.2022.146502

Cited by 10 publications

(4 citation statements)

References 43 publications

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“…Accurate normalization of data is necessary to avoid erroneous differences in target gene expression (31)(32)(33). In this study, we identified suitable reference genes for Gardnerella swidsinskii in media supplemented with 1% maltotriose or glycogen.…”

Section: Discussionmentioning

confidence: 99%

Investigation of expression of genes encoding glycogen degrading enzymes inGardnerella swidsinskiiand identification of reference genes for quantitative real-time PCR

Kim,

Fernando,

Kularatne

et al. 2024

Preprint

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Gardnerellaspp. express and export enzymes for the breakdown of glycogen into glucose, maltose, and malto-oligosaccharides for consumption by the vaginal microbiota but how the expression of these "public goods" is affected by substrate and product levels in the environment is not known. Accurate measurement of relative gene expression using real-time quantitative PCR relies on the identification of appropriate reference genes whose expression levels remain constant under the conditions of the study. Currently, no reference genes have been identified for gene expression analysis ofGardnerellaspp. The objectives of this study were to identify reference genes and apply them in determining the relative gene expression levels of genes encoding α-amylase and α-amylase-pullulanase in media supplemented with substrate (glycogen) or a preferred product (maltotriose). Ten candidate reference genes were evaluated and analysis of Cq values from qPCR using multiple algorithms identified uppS (encoding polyprenyl diphosphate synthase) as the top comprehensively ranked reference gene followed by gatA (encoding Asp-tRNA/Glu-tRNA amidotransferase subunit gatA). Interpretation of the Cq values for α-amylase and α-amylase-pullulanase was performed by applying these two reference genes in the calculation of relative gene expression levels. α-amylase-pullulanase gene expression was upregulated in media supplemented with 1% glycogen in comparison to media supplemented with 1% maltotriose suggesting a regulatory mechanism inG. swidsinskiithat responds to nutrient availability. No significant difference in gene expression of α-amylase was observed suggesting expression is not influenced by substrate availability. The RNA purification protocol and reference genes validated in this study will be useful in future studies of gene expression inGardnerella.

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Section: Discussionmentioning

confidence: 99%

Investigation of expression of genes encoding glycogen degrading enzymes inGardnerella swidsinskiiand identification of reference genes for quantitative real-time PCR

Kim,

Fernando,

Kularatne

et al. 2024

Preprint

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“…When it comes to samples in total stages, including stage 1, stage 2, and stage 3, EIF3K demonstrated the best stability. EIF3K (Eukaryotic translation initiation factor 3) has been suggested as one of the most stably expressed HKGs in bovine endometrium [ 25 ]. The uterus caruncle is the pitting structure of endometrium, so our results may partly validate previous research.…”

Section: Discussionmentioning

confidence: 99%

Screening of Candidate Housekeeping Genes in Uterus Caruncle by RNA-Sequence and qPCR Analyses in Different Stages of Goat (Capra hircus)

Zhou

Zhang

et al. 2023

Animals

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The uterus is a critical pregnancy organ for mammals. The normal growth and development of ruminant uterus caruncles are crucial to maintain gestation and fetal health in goats. Quantitative real-time polymerase chain reaction (qRT-PCR) is a reliable tool to study gene expression profiling for exploring the intrinsic mechanism underlying the conversion process of uterus caruncle tissue. However, the candidate housekeeping genes (HKGs) are required for normalizing the expression of function genes. In our study, 22 HKGs were selected from analyzing transcriptome data at non-pregnancy and pregnancy processes and previous reports about HKGs in goat tissues. We assessed them for expression suitability in 24 samples from uterus tissues at 15 non-pregnant days (Stage 1), early (Stage 2), and medium-later pregnant days (Stage 3). The expression stability of these genes was evaluated by using geNorm, Normfinder, Bestkeeper, and Delta Ct algorithms and, comprehensively, by ReFinder. In addition, the most and least stable HKGs were used to normalize the target genes expression of SPP1, VEGFA, and PAG8. It was found that traditional reference genes, such as ACTB and GAPDH, were not suitable for target gene normalization. In contrast, PPIB selected from RNA sequencing data and EIF3K selected from previous references showed the least variation and were recommended as the best HKGs during the nonpregnant stage and the whole stages of goat uterus caruncle tissue, respectively. It is the first time the HKGs genes in uterus during the non-pregnant day and throughout the total pregnancy have been explored. These findings found suitable HKGs in uterus caruncle tissues at various stages of non-pregnancy and pregnancy; these can be useful for gene expression studies to reveal the molecular mechanisms of uterus development in goats.

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“…Primer specificity was checked by sequence alignment using Nucleotide BLAST at the NCBI genome browser gateway, and was further confirmed in a standard PCR reaction followed by ethidium-bromide staining on 2% agarose gel as previously described ( Shandilya et al., 2021 ). The reference genes glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) ( Sharma et al., 2019 ) and ubiquitously expressed prefoldin like chaperone ( UXT ) ( Wang et al., 2022 ) were used as the internal control to normalize the expression of the target gene transcript levels. A total of 4 reference genes ( GAPDH, UXT, β-actin and β2-microglobulin) were tested as internal control genes.…”

Section: Methodsmentioning

confidence: 99%

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function

Xu¹,

Shandilya²,

Yiannikouris³

et al. 2023

Animal Nutrition

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Analysis of stability of reference genes for qPCR in bovine preadipocytes during proliferation and differentiation in vitro

Cited by 10 publications

References 43 publications

Investigation of expression of genes encoding glycogen degrading enzymes inGardnerella swidsinskiiand identification of reference genes for quantitative real-time PCR

Investigation of expression of genes encoding glycogen degrading enzymes inGardnerella swidsinskiiand identification of reference genes for quantitative real-time PCR

Screening of Candidate Housekeeping Genes in Uterus Caruncle by RNA-Sequence and qPCR Analyses in Different Stages of Goat (Capra hircus)

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function

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