title: A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells.
abstract: As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted.
authors.affiliation: Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. alv@sund.ku.dk.
authors.forename: Anna Lindeløv
authors.initials: AL
authors.lastname: Vestergaard
authors.affiliation: Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. m.blankestijn@umcg.nl.
authors.forename: Maaike
authors.initials: M
authors.lastname: Blankestijn
authors.affiliation: Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. jonathan@sund.ku.dk.
authors.forename: Jonathan Lucien
authors.initials: JL
authors.lastname: Stahl
authors.affiliation: Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. epallesen@sund.ku.dk.
authors.forename: Emil Marek Heymans
authors.initials: EM
authors.lastname: Pallesen
authors.affiliation: Center for Non-coding RNA in Technology and Health, Department of Pediatrics, Herlev and Gentofte Hospital, 2730 Herlev, Denmark. cbberthelsen@gmail.com.
authors.forename: Claus Heiner
authors.initials: CH
authors.lastname: Bang-Berthelsen
authors.affiliation: Center for Non-coding RNA in Technology and Health, Department of Pediatrics, Herlev and Gentofte Hospital, 2730 Herlev, Denmark. flemming.pociot.01@regionh.dk.|Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. flemming.pociot.01@regionh.dk.
authors.forename: Flemming
authors.initials: F
authors.lastname: Pociot
authors.affiliation: Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. guy_novotny@runbox.com.
authors.forename: Guy Wayne
authors.initials: GW
authors.lastname: Novotny
authors.affiliation: Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. lundh@sund.ku.dk.
authors.forename: Morten
authors.initials: M
authors.lastname: Lundh
authors.affiliation: Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. tmpo@sund.ku.dk.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mandrup-Poulsen
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics*; D020871:RNA Stability; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D059467:Transcriptome
chemical_list: D035683:MicroRNAs
keywords: method; microRNA; normalization; pancreas; purification; qPCR; quantification