Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR

Zampieri, Denise; Nora, Luísa Czamanski; Basso, Vanessa; Camassola, Marli; Dillon, Aldo José Pinheiro

doi:10.1007/s00294-014-0421-6

Cited by 22 publications

(16 citation statements)

References 24 publications

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“…Quantification of miRNAs and mRNA using quantitative real-time PCR Quantification of miR-221 and miR-222 in the G418-selected clones was performed by using SYBR Premix Ex Taq II (Perfect Real Time) assays (Applied TaKaRa Biosystems), followed by detection with the CFX96TM PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions (Technote 5859). The real-time quantitative PCR reaction conditions were as follows: denaturation at 95 1C for 5 min, followed by 40 cycles of denaturation at 95 1C for 10 s and annealing and extension at 60 1C for 30 s. 36 The U6 small nuclear RNA (Guanzhou Ribobio, Guanzhou, China) was used to normalize the relative abundance of miR-221 and miR-222. Data shown represent three independent experiments performed in triplicate.…”

Section: Rna Extractionmentioning

confidence: 99%

Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3

et al. 2014

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Aberrantly expressed microRNAs (miRNAs) are involved in breast tumorigenesis. It is still unclear if and how miRNAs-221/222 are implicated in breast cancer and the resistance to estrogen receptor modulator tamoxifen. We investigated the roles and mechanisms of miR-221/222 in breast cancer cells, particularly in modulating response to tamoxifen therapy. MCF-7 and MDA-MB-231 breast cancer cells were transfected with antisense oligonucleotides AS-miR-221 and AS-miR-222 and their expression of miR-221 and miR-222 was assessed. The correlation of miR-221/222 with tissue inhibitor of metalloproteinase-3 (TIMP3) expression was investigated by fluorescence quantitative PCR and western blotting analysis. The therapeutic sensitivity of these cells, transfected and untransfected, to tamoxifen was determined. Transfection of AS-miR-221 and AS-miR-222 dramatically inhibited expression of miR-221 and miR-222, respectively, in both MCF-7 and MDA-MB-231 cells (P<0.05-0.01). Downregulation of miR-221/222 significantly increased the expression of TIMP3 compared with controls (P<0.05-0.01). The viability of estrogen receptor (ER)-positive MCF-7 cells transfected with AS-miR-221 or/and AS-miR-222 was significantly reduced by tamoxifen (P<0.05-0.01). We have demonstrated for the first time that suppression of miRNA-221/222 increases the sensitivity of ER-positive MCF-7 breast cancer cells to tamoxifen. This effect is mediated through upregulation of TIMP3. These findings suggest that upregulation of TIMP3 via inhibition of miRNA-221/222 could be a promising therapeutic approach for breast cancer.

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Section: Rna Extractionmentioning

confidence: 99%

Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3

et al. 2014

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“…A large number of research papers have been published on reference genes under different stresses or from different organs in plants (Warzybok and Migocka 2013; Lin et al 2014), and similar works in filamentous fungi are gradually being conducted (Zampieri et al 2014; Zhou et al 2011). It is striking that most of the applied reference genes for fungi were directly copied from the existing results in plants or animals, such as actin , tubulin and 18S rRNA (Fang and Bidochka 2006; Yan and Liou 2006).…”

Section: Introductionmentioning

confidence: 99%

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

Zhang

Hou

et al. 2017

AMB Expr

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Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek–Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.

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“…www.nature.com/scientificreports www.nature.com/scientificreports/ to the findings of Anita et al 10 . ACT1, TUB1, and SDHA are also commonly utilized reference genes in stability analysis and target gene expression studies 10,17,[34][35][36]38 . A study by Li et al clearly demonstrated the unsuitability of ACT1, TUB1, and SDHA as reference genes for inducible expression analysis in C. glabrata cells treated with fluconazole 17 .…”

Section: Discussionmentioning

confidence: 99%

Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment

Paul

Chakrabarti

Rudramurthy

et al. 2020

Sci Rep

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Candida tropicalis arises as one of the predominant non-Candida albicans Candida (ncAc) species causing invasive candidiasis in Asian countries. A rise in reports of C. tropicalis with a parallel increase in fluconazole resistance has also been observed. The genes and underlying pathways associated with azole antifungal resistance in C. tropicalis is still not properly understood. the Rt-qpcR is the most promising approach for expression analysis of target genes to understand the mechanisms of resistance. the reliability and reproducibility of this technique depend on the selection of suitable reference genes for the normalization in expression study. the present study investigated the expression stability levels of ten genes including ACT1, EF1, GAPDH, PGK1, RDN5.8, RDN18, RDN28, SDHA, TUB1, and UBC13 for their suitability in fluconazole treated/untreated C. tropicalis. the stability levels of these genes were examined by the ∆∆ct, ΔCT, Pfaffl methods and five independent software including hkgfinder, genorm, normfinder, BestKeeper, and Reffinder software. We report, the EF1 and ACT1 were the most stable reference genes for normalization and can be used for the gene expression analysis in C. tropicalis. To the best of our knowledge, our study is the first to select and validate the reference genes in C. tropicalis for Rt-qpcR based expression analysis. Candida tropicalis, a non-Candida albicans Candida (NCAC) resides in human skin, genitourinary, respiratory, and gastrointestinal tracts as a part of the normal microbiota 1-3. In Asian countries, C. tropicalis has emerged as the predominant NCAC species causing invasive candidiasis (IC), particularly candidemia 4-6. Fluconazole is the most common antifungal drug used to treat candidemia due to C. tropicalis. The rise in IC due to C. tropicalis has been paralleled with an increase in fluconazole resistance, especially in Asian countries 4-6. The differential expression of ergosterol biosynthesis pathway genes, ATP-binding cassette (ABC), and major facilitator superfamily (MFS) drug transporters are directly linked with the azole resistance in C. tropicalis 7-9. Although various studies demonstrated the role of these mechanisms in azole resistant C. tropicalis, the principle pathways and regulatory circuits implicated are yet to be elucidated. Profiling of gene expression is a powerful approach to determine the pattern response to various stimuli including drugs and gives a holistic impression of cellular function in any living cell 10. Usually, gene expression can be estimated by multiple methods, including RNase protection assay, Northern blotting, real-time quantitative PCR (RT-qPCR), and semi-quantitative reverse-transcription PCR 11. RT-qPCR has received special attention due to its significantly higher accuracy, sensitivity, and rapidity allowing high throughput results, detection of mRNAs with low-abundance 12 and mRNA copy number measurement 13. As a result, RT-qPCR platform has been utilized for diverse applications including gene expression analysis 11,14...

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Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR

Cited by 22 publications

References 24 publications

Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3

Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment

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