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

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          Abstract

          The mean expression value: a new method for accurate and reliable normalization of microRNA expression data from RT-qPCR experiments.

          Abstract

          Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.

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          Widespread microRNA repression by Myc contributes to tumorigenesis.

          Tsung-Cheng Chang, Duonan Yu, Yun-Sil Lee … (2008)
          The c-Myc oncogenic transcription factor (Myc) is pathologically activated in many human malignancies. Myc is known to directly upregulate a pro-tumorigenic group of microRNAs (miRNAs) known as the miR-17-92 cluster. Through the analysis of human and mouse models of B cell lymphoma, we show here that Myc regulates a much broader set of miRNAs than previously anticipated. Unexpectedly, the predominant consequence of activation of Myc is widespread repression of miRNA expression. Chromatin immunoprecipitation reveals that much of this repression is likely to be a direct result of Myc binding to miRNA promoters. We further show that enforced expression of repressed miRNAs diminishes the tumorigenic potential of lymphoma cells. These results demonstrate that extensive reprogramming of the miRNA transcriptome by Myc contributes to tumorigenesis.
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            Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues.

            Heidi J Peltier, Gary J Latham (2008)
            Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.
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              An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues.

              C-G Liu, G Calin, B. Meloon … (2004)
              MicroRNAs (miRNAs) are a class of small noncoding RNA genes recently found to be abnormally expressed in several types of cancer. Here, we describe a recently developed methodology for miRNA gene expression profiling based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes. We used these microarrays to obtain highly reproducible results that revealed tissue-specific miRNA expression signatures, data that were confirmed by assessment of expression by Northern blots, real-time RT-PCR, and literature search. The microchip oligolibrary can be expanded to include an increasing number of miRNAs discovered in various species and is useful for the analysis of normal and disease states.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Genome Biol
                Title: Genome Biology
                Publisher: BioMed Central
                ISSN (Print): 1465-6906
                ISSN (Electronic): 1465-6914
                Publication date (Print): 2009
                Publication date (Electronic): 16 June 2009
                Volume: 10
                Issue: 6
                Page: R64
                Affiliations
                [1 ]Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, Ghent, Belgium
                [2 ]Department of Tumour Genetics, German Cancer Center, Im Neuenheimer Feld 280, Heidelberg, Germany
                Article
                Publisher ID: gb-2009-10-6-r64
                DOI: 10.1186/gb-2009-10-6-r64
                PMC ID: 2718498
                PubMed ID: 19531210
                SO-VID: 182c5058-d8f8-4525-99d5-2bcf2d2a2d08
                Copyright © Copyright © 2009 Mestdagh et al.; licensee BioMed Central Ltd.
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 2 April 2009
                Date revision received : 2 April 2009
                Date accepted : 16 June 2009
                Categories
                Subject: Method

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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