Direct and sensitive miRNA profiling from low-input total RNA

Wang, Hui; Ach, Robert A.; Curry, Bo

doi:10.1261/rna.234507

Cited by 279 publications

(244 citation statements)

References 30 publications

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“…The concentration and quality of the isolated RNA was analysed using the Tecan infinite M200 plate reader and the determined concentration used for normalizing the RNA input in down‐stream steps. The labelling reaction was based on a method by Wang et al [2007]. A total of 300 ng RNA was used in the labelling reaction, consisting of synthetic spike‐in controls, phosphate‐cytidyl‐uridyl‐DY547‐3′ RNA dinucleotides (50 μM, Thermo Fisher Scientific), ATP (1 mM), Tris‐HCl (pH 7.8, 50 mM), MgCl 2 (10 mM), DTT (1 mM), bovine serum albumin (BSA, 10 μg mL −1 ), DMSO (25% (v/v)), and T4 ligase (20 U, New England Biolabs).…”

Section: Methodsmentioning

confidence: 99%

N^ϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

Holik

Lieder

Kretschy

et al. 2016

J of Cellular Biochemistry

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Advanced glycation endproducts, formed in vivo, but also by the Maillard reaction upon thermal treatment of foods, have been associated with the progression of pathological conditions such as diabetes mellitus. In addition to the accumulation with age, exogenous AGEs are introduced into the circulation from dietary sources. In this study, we investigated the effects of addition of free Nϵ‐carboxymethyllysine (CML), a well‐characterized product of the Maillard reaction, on adipogenesis in 3T3‐L1 preadipocytes. Treatment with 5, 50, or 500 μM CML resulted in increased lipid accumulation to similar extents, by 11.5 ± 12.6%, 12.9 ± 8.6%, and 12.8 ± 8.5%, respectively. Long‐term treatment with 500 μM CML during adipogenesis resulted in increases in miR‐103 and miR‐143 levels, two miRNAs described to be involved in impaired glucose homeostasis and increased lipid accumulation. Furthermore, the expression of genes associated with these miRNAs, consisting of Akt1, PI3k, and Cav1 was regulated by CML. Short‐term treatment of mature 3T3‐L1 adipocytes with CML resulted in decreased basal glucose uptake. These results, indicate that the addition of protein‐free CML to 3T3‐L1 cells influence parameters associated with adipogenesis and glucose homeostasis at transcriptional, and functional level; this indicates that free CML derived from exogenous sources, in addition to protein‐bound CML may be relevant in this context. J. Cell. Biochem. 117: 2413–2422, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

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Section: Methodsmentioning

confidence: 99%

N^ϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

Holik

Lieder

Kretschy

et al. 2016

J of Cellular Biochemistry

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“…Both the short length and the heterogeneous GC content of miRNAs result in large impede intervals of melting temperatures complicating optimized probe or primer design (Benes and Castoldi 2010;Yin et al 2008). Extended loop probes (Wang et al 2007), stem-loop primer (Chen et al 2005), or the incorporation of modified nucleotides, e.g. locked nucleic acids (Castoldi et al 2008) or 2'-O-(2-methoxyethyl)-derivatives (Beuvink et al 2007) have been used for normalization of melting temperatures.…”

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confidence: 99%

Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity?

2010

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To cite this version:Swanhild U. Meyer, Michael W. Pfaffl, Susanne E. Ulbrich. Normalization strategies for microRNA profiling experiments: a 'normal' way to a hidden layer of complexity?. Biotechnology Letters, Springer Verlag, 2010, 32 (12) review provides a critical overview of commonly used and newly developed normalization methods for miRNA RT-qPCR, miRNA hybridization microarray, and small RNA-seq datasets. Emphasis is laid on the complexity, the importance and the potential for further optimization of normalization techniques for miRNA profiling datasets.

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“…The RNA samples were labeled and processed according to manufacturer's recommended protocols (Wang et al 2007). In brief, 100 ng of total RNA was dephosphorylated with calf intestinal alkaline phosphatase, followed by denaturing with heat in the presence of dimethyl sulfoxide (DMSO).…”

Section: Mirnas Microarray Profilingmentioning

confidence: 99%

MicroRNA Profiling in Great Saphenous Vein Tissues of Patients with Chronic Venous Insufficiency

Cui

Guang

Huang

et al. 2012

Tohoku J. Exp. Med.

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Chronic venous insufficiency (CVI) is a common disease characterized by structural and functional abnormalities of the venous system. Until recently, the pathogenesis of CVI remains largely unknown. MicroRNAs (miRNAs) are a family of endogenous small non-coding RNAs emerged as post-transcriptional gene repressors and play essential roles in diverse pathological processes including vascular disease. However, their roles in CVI have not been elucidated. In this study, we employed oligonucleotide microarrays to perform a genome-wide miRNAs profiling in the great saphenous vein (GSV) tissues of patients with CVI. Our results revealed a total of 14 miRNAs that are expressed differentially in GSV tissues. Among them nine miRNAs were found significantly up-regulated, while five miRNAs were downregulated significantly. Real-time RT-PCR verified statistically consistent expression of three selected miRNAs (miR-34a, miR-155 and miR-202) with microarrays analysis. These three miRNAs, which were described as crucial regulators in many biological processes and vascular diseases, might also play important roles in CVI. Functional annotation of target genes of differentially expressed miRNAs via bioinformatics approaches revealed that these predicted targets were significantly enriched and involved in several key signaling pathways important for CVI, including mitogen-activated protein kinase pathways, pathways in cancer, apoptosis, and cell cycle, and p53 signaling pathways. In summary, miRNAs might involve in multiple signaling pathways contributing to the pathological processes of CVI. These data may provide fundamental insights into the molecular basis of CVI, which may aid in designing novel approaches for prevention and treatment of this complex disease.Keywords: bioinformatics; chronic venous insufficiency; great saphenous vein; microarray; microRNA Tohoku J. Exp. Med., 2012 Dec, 228 (4), 341-350. © 2012 Tohoku University Medical Press Chronic venous insufficiency (CVI) is a complex medical condition characterized by structural and functional abnormalities of the venous system, which is commonly seen in humans. CVI is manifested with a range of signs, including varicose veins, trophic skin changes and venous ulcers. It has a considerable socioeconomic impact in many countries due to its high prevalence, cost of investigations and management, deterioration of the quality of life and loss of working days (Rabe and Pannier 2010). To date, in spite of numerous studies on CVI, the etiology and pathogenesis of CVI remains largely unknown.MicroRNAs (miRNAs) are a family of highly conserved, endogenous small non-coding RNAs (19-24nt) that post-transcriptionally repress gene expression via degradation or translational inhibition of their target mRNAs. They play important roles in nearly all physiological and pathological processes, including cell differentiation, proliferation, apoptosis, cardiovascular disease, and human cancers (Bartel 2004). There is mounting evidence suggesting that miRNAs had distinct expression profiles an...

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Direct and sensitive miRNA profiling from low-input total RNA

Cited by 279 publications

References 30 publications

N^ϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

N^ϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity?

MicroRNA Profiling in Great Saphenous Vein Tissues of Patients with Chronic Venous Insufficiency

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Direct and sensitive miRNA profiling from low-input total RNA

Cited by 279 publications

References 30 publications

Nϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

Nϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity?

MicroRNA Profiling in Great Saphenous Vein Tissues of Patients with Chronic Venous Insufficiency

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N^ϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

N^ϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells