A novel and universal method for microRNA RT-qPCR data normalization

Mestdagh, Pieter; Vlierberghe, Pieter Van; Weer, An-Sofie De; Muth, Daniel; Westermann, Frank; Speleman, Frank; Vandesompele, Jo

doi:10.1186/gb-2009-10-6-r64

Cited by 908 publications

(859 citation statements)

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“…Data from cell lines were normalized to U44 RNA expression. miRNA expression in primary tumor samples was analysed as previously described, 33 and normalization was performed by using a set of miRNAs stably expressed in neuroblastomas. Real-time RT-PCR was performed by using the Applied Biosystems 7000 Sequence Detection system (Applied Biosystems, Foster City, CA, USA).…”

Section: Discussionmentioning

confidence: 99%

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival

et al. 2011

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Several microRNA (miRNA) loci are found within genomic regions frequently deleted in primary neuroblastoma, including miR-885-5p at 3p25.3. In this study, we demonstrate that miR-885-5p is downregulated on loss of 3p25.3 region in neuroblastoma. Experimentally enforced miR-885-5p expression in neuroblastoma cell lines inhibits proliferation triggering cell cycle arrest, senescence and/or apoptosis. miR-885-5p leads to the accumulation of p53 protein and activates the p53 pathway, resulting in upregulation of p53 targets. Enforced miR-885-5p expression consistently leads to downregulation of cyclin-dependent kinase (CDK2) and mini-chromosome maintenance protein (MCM5). Both genes are targeted by miR-885-5p via predicted binding sites within the 3 0 -untranslated regions (UTRs) of CDK2 and MCM5. Transcript profiling after miR-885-5p introduction in neuroblastoma cells reveals alterations in expression of multiple genes, including several p53 target genes and a number of factors involved in p53 pathway activity. Taken together, these data provide evidence that miR-885-5p has a tumor suppressive role in neuroblastoma interfering with cell cycle progression and cell survival. Cell Death and Differentiation (2011) 18, 974-984; doi:10.1038/cdd.2010.164; published online 14 January 2011MicroRNAs (miRNAs) are non-coding short (18-24 nt) RNAs, controlling gene expression post trancriptionally, via inhibiting translation or triggering degradation of multiple target mRNAs. They are vitally important regulators of proliferation, differentiation and apoptosis. Upregulated and downregulated miRNAs have been found in different malignancies, implicating miRNAs as oncogenes or tumor suppressors. 1 The increasing body of evidence for miRNA involvement in tumorigenesis has prompted the search in cancer-associated genomic regions for miRNA loci. 2 In a deletion region at 13q14, miR-15 and miR-16, are downregulated in B-cell chronic lymphocytic leukemia. Both miR-15 and miR-16 negatively regulate BCL2 and positively regulate apoptosis, which could explain their frequent inactivation in leukemia. 3 Later, miR-34a at 1p36 emerged as a tumor suppressor gene (TSG). Several independent studies demonstrated anti-proliferative and pro-apoptotic potential of miR-34a. 4,5 The potential for involvement of as yet unreported miRNAs in cancer pathogenesis is great.Neural crest-derived neuroblastoma, one of the most common solid tumors in children, is frequently characterized as an 'enigmatic neoplasm'. Neuroblastoma has the highest rate of spontaneous regression of all human malignancies, however, B40% of neuroblastomas rapidly progress despite multimodal treatment regimes. 6,7 TP53 mutations are rare in neuroblastoma, which very often retain functional p53, suggesting that inhibitors of the p53 pathway or loss of p53 pathway positive regulators may be involved in neutralizing p53 activity. 8,9 Neuroblastomas with poor outcome are subdivided into at least two biologically distinct groups, either with or without amplification of the MYCN oncogene....

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Section: Discussionmentioning

confidence: 99%

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival

et al. 2011

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“…The expression levels of miRNAs in all groups were calculated using the comparative cycle threshold (C T ) method, whereby a lower C T value is consistent with faster detection of fluorescence and therefore higher expression, and SDS software v2.3 using automatic baseline settings and a threshold of 0.2. Samples were normalized by the mean of expressed miRNAs, as described, 27 whereby C T values ≥ 35 were considered to be below the detection level of the assay and therefore only miRNAs with a C T value o 35 were included in the analyses. The mean miRNA C T value was then calculated for each group and subtracted from each 28 where comparative C T (ΔΔC T ) is defined as the difference between ΔC T of comparison groups (eg, (ΔC T P+M) − (ΔC T P − M)).…”

Section: Assessment Of Mirna Expression In Discovery Cohort From Poolmentioning

confidence: 99%

microRNA-10b is a prognostic biomarker for melanoma

et al. 2016

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Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n = 79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P = 0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (Po 0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P = 0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P = 0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.

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“…All miRNAs with cycle threshold (Ct) values .35 were considered nonamplified or not expressed and were excluded from analysis. Mean normalization was performed by subtracting the mean sample Ct from the individual miRNA Ct values (19). Relative quantification of gene expression was determined using the comparative Ct method [2 (2DDCt) ] as previously described (20).…”

Section: Mirna Expression Profiling In Cf Bronchial Brushingsmentioning

confidence: 99%