A simple method to assess group difference in RT-qPCR reference gene selection using GeNorm: The case of the placental sex

St‐Pierre, Joey; Grégoire, Jean-Charles; Vaillancourt, Cathy

doi:10.1038/s41598-017-16916-y

Cited by 27 publications

(17 citation statements)

References 22 publications

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“…Quantitative RT-PCR analysis was carried out as per the manufacturer’s instructions (Applied Biosystems, Foster City, CA, United States) using Taq-Man Gene Expression Assays on a Qiagen Rotor-Gene Q. Data were normalized to vehicle treated controls and compared to five reference targets (Hmbs, B2M, TBP, H2afv, and 18s), which were evaluated for expression stability using GeNorm ( St-Pierre et al, 2017 ).…”

Section: Methodsmentioning

confidence: 99%

The Acute Effects of 5 Fluorouracil on Skeletal Muscle Resident and Infiltrating Immune Cells in Mice

et al. 2020

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5 fluorouracil (5FU) has been a first-choice chemotherapy drug for several cancer types (e.g., colon, breast, head, and neck); however, its efficacy is diminished by patient acquired resistance and pervasive side effects. Leukopenia is a hallmark of 5FU; however, the impact of 5FU-induced leukopenia on healthy tissue is only becoming unearthed. Recently, skeletal muscle has been shown to be impacted by 5FU in clinical and preclinical settings and weakness and fatigue remain among the most consistent complaints in cancer patients undergoing chemotherapy. Monocytes, or more specifically macrophages, are the predominate immune cell in skeletal muscle which regulate turnover and homeostasis through removal of damaged or old materials as well as coordinate skeletal muscle repair and remodeling. Whether 5FU-induced leukopenia extends beyond circulation to impact resident and infiltrating skeletal muscle immune cells has not been examined. The purpose of the study was to examine the acute effects of 5FU on resident and infiltrating skeletal muscle monocytes and inflammatory mediators. Male C57BL/6 mice were given a physiologically translatable dose (35 mg/kg) of 5FU, or PBS, i.p. once daily for 5 days to recapitulate 1 dosing cycle. Our results demonstrate that 5FU reduced circulating leukocytes, erythrocytes, and thrombocytes while inducing significant body weight loss (>5%). Flow cytometry analysis of the skeletal muscle indicated a reduction in total CD45+ immune cells with a corresponding decrease in total CD45+CD11b+ monocytes. There was a strong relationship between circulating leukocytes and skeletal muscle CD45+ immune cells. Skeletal muscle Ly6cHigh activated monocytes and M1-like macrophages were reduced with 5FU treatment while total M2-like CD206+CD11c- macrophages were unchanged. Interestingly, 5FU reduced bone marrow CD45+ immune cells and CD45+CD11b+ monocytes. Our results demonstrate that 5FU induced body weight loss and decreased skeletal muscle CD45+ immune cells in association with a reduction in infiltrating Ly6cHigh monocytes. Interestingly, the loss of skeletal muscle immune cells occurred with bone marrow cell cycle arrest. Together our results highlight that skeletal muscle is sensitive to 5FU’s off-target effects which disrupts both circulating and skeletal muscle immune cells.

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Section: Methodsmentioning

confidence: 99%

The Acute Effects of 5 Fluorouracil on Skeletal Muscle Resident and Infiltrating Immune Cells in Mice

et al. 2020

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“…A set of three endogenous reference miRNAs were identified using geNorm and NormFinder ( 30 , 31 ). The geometric mean of the three normalizers was used to normalize the miRNA expression data across subjects.…”

Section: Methodsmentioning

confidence: 99%

Downregulation of the let‑7 family of microRNAs may promote insulin receptor/insulin‑like growth factor signalling pathways in pancreatic ductal adenocarcinoma

Nweke

Brand

2020

Oncol Lett

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Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type characterized by dysregulated cell signalling pathways and resistance to treatment. The insulin-like growth factor (IGF) signalling pathway has been identified to have a role in tumour progression and therapy resistance. However, its regulatory roles in PDAC have remained to be fully elucidated. In the present study, dysregulated microRNAs (miRNAs) in PDAC were explored with a focus on those that may be involved in regulating the insulin/IGF signalling pathway. A total of 208 patients were recruited, comprising 112 patients with PDAC, 50 patients with chronic pancreatitis (CP) and 46 subjects as a control group (CG). miRNA-specific quantitative PCR assays were used to measure 300 candidate miRNAs. The Student's t-test was applied to compare miRNA regulation between cancer patients and controls with a false discovery rate correction using Bonferroni-type comparison procedures. The DIANA-mirPath v.3 tool and HMDD v3.0 were used to identify miRNA-mRNA interactions within specific pathways. In patients with PDAC, 42 miRNAs were significantly upregulated and 42 were downregulated compared to the CG (P<0.01). In the PDAC vs. CP analysis, 16 significantly (P<0.01) upregulated and 16 downregulated miRNAs were identified. Of note, members of the let-7 family of miRNAs were downregulated and were indicated to target several components of the insulin receptor (INSR)/IGF pathway, including receptors and binding proteins, for upregulation and thus, may enable the activation of the pathway. Downregulation of the let-7 family may help promote the INSR/IGF pathway in PDAC. It may thus be an effective target for the development of INSR/IGF pathway-specific treatment strategies.

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“…Five different statistical algorithms were used to evaluate the stabilities of candidate reference gene expression. The geNorm algorithm was used to calculate the gene expression stability by measuring the M values, and to determine the optimal number of reference genes for this study as described previously [27]. The NormFinder algorithm utilized a linear mixed effect model to estimate both intra- and inter-group variations, and to rank the candidate reference genes by the stabilityvalues (SV) [28].…”

Section: Methodsmentioning

confidence: 99%

Determination of Suitable RT-qPCR Reference Genes for Studies of Gene Functions in Laodelphax striatellus (Fallén)

Liu

Dong

et al. 2019

Genes

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The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus. The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 (LsRPL5) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin (Lsα-TUB) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus. LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus, while beta-tubulin (Lsβ-TUB) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1-silenced L. striatellus. The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus. The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus.

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A simple method to assess group difference in RT-qPCR reference gene selection using GeNorm: The case of the placental sex

Cited by 27 publications

References 22 publications

The Acute Effects of 5 Fluorouracil on Skeletal Muscle Resident and Infiltrating Immune Cells in Mice

The Acute Effects of 5 Fluorouracil on Skeletal Muscle Resident and Infiltrating Immune Cells in Mice

Downregulation of the let‑7 family of microRNAs may promote insulin receptor/insulin‑like growth factor signalling pathways in pancreatic ductal adenocarcinoma

Determination of Suitable RT-qPCR Reference Genes for Studies of Gene Functions in Laodelphax striatellus (Fallén)

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