title: Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR.
abstract: The high-throughput analysis of microRNAs (miRNAs) circulating within the blood of healthy and diseased individuals is an active area of biomarker research. Whereas quantitative real-time reverse transcription polymerase chain reaction (qPCR)-based methods are widely used, it is yet unresolved how the data should be normalized. Here, we show that a combination of different algorithms results in the identification of candidate reference miRNAs that can be exploited as normalizers, in both discovery and validation phases. Using the methodology considered here, we identify normalizers that are able to reduce nonbiological variation in the data and we present several case studies, to illustrate the relevance in the context of physiological or pathological scenarios. In conclusion, the discovery of stable reference miRNAs from high-throughput studies allows appropriate normalization of focused qPCR assays.
authors.forename: Francesco
authors.initials: F
authors.lastname: Marabita
authors.forename: Paola
authors.initials: P
authors.lastname: de Candia
authors.forename: Anna
authors.initials: A
authors.lastname: Torri
authors.forename: Jesper
authors.initials: J
authors.lastname: Tegnér
authors.forename: Sergio
authors.initials: S
authors.lastname: Abrignani
authors.forename: Riccardo L
authors.initials: RL
authors.lastname: Rossi
mesh_terms: D000465:Algorithms; D015415:Biomarkers / Q000097:blood; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics* / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D035683:MicroRNAs
keywords: Normfinder; circulating miRNA; geNorm; normalization; qPCR; reference genes