title: Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy.
abstract: Small nucleolar RNAs (snoRNAs) have been used for normalization in glomerular microRNA (miRNA) quantification without confirmation of validity. Our aim was to identify glomerular reference miRNAs in IgA nephropathy. We compared miRNAs in human paraffin-embedded renal biopsies from patients with cellular-crescentic IgA-GN (n = 5; crescentic IgA-GN) and non-crescentic IgA-GN (n = 5; IgA-GN) to mild interstitial nephritis without glomerular abnormalities (controls, n = 5). Laser-microdissected glomeruli were used for expression profiling of 762 miRNAs by low-density TaqMan arrays (cards A and B). The comparison of different normalization methods (GeNormPlus, NormFinder, global mean and snoRNAs) in crescentic IgA-GN, IgA-GN and controls yielded similar results. However, levels of significance and the range of relative expression differed. In median, two normalization methods demonstrated similar results. GeNormPlus and NormFinder gave different top ranked reference miRNAs. Stability ranking for snoRNAs varied between cards A and B. In conclusion, we suggest the geometric mean of the most stable reference miRNAs found in GeNormPlus (miR-26b-5p), NormFinder (miR-28-5p) and snoRNAs (RNU44) as reference. It should be considered that significant differences could be missed using one particular normalization method. As a starting point for glomerular miRNA studies in IgA nephropathy we provide a library of miRNAs.
authors.affiliation: Department of Nephropathology, Friedrich Alexander University (FAU) Erlangen-Nuremberg, Erlangen, Germany.|Institute of Pathology, Hannover Medical School, Hannover, Germany.
authors.forename: Clemens L
authors.initials: CL
authors.lastname: Bockmeyer
authors.affiliation: Institute of Pathology, University Hospital of Cologne, Cologne, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Säuberlich
authors.affiliation: Institute of Pathology, University Hospital of Cologne, Cologne, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Wittig
authors.affiliation: Institute of Pathology, University Hospital of Cologne, Cologne, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Eßer
authors.affiliation: Department of Nephropathology, Friedrich Alexander University (FAU) Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Sebastian S
authors.initials: SS
authors.lastname: Roeder
authors.affiliation: Children's Hospital, Pediatrics II, University of Duisburg-Essen, Essen, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Vester
authors.affiliation: Children's Hospital, Pediatrics II, University of Duisburg-Essen, Essen, Germany.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Hoyer
authors.affiliation: Department of Nephrology, Hannover Medical School, Hannover, Germany.
authors.forename: Putri A
authors.initials: PA
authors.lastname: Agustian
authors.affiliation: Institute of Pathology, University Hospital of Cologne, Cologne, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Zeuschner
authors.affiliation: Department of Nephropathology, Friedrich Alexander University (FAU) Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Amann
authors.affiliation: Department of Nephropathology, Friedrich Alexander University (FAU) Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Daniel
authors.affiliation: Institute of Pathology, University Hospital of Cologne, Cologne, Germany.
authors.forename: Jan U
authors.initials: JU
authors.lastname: Becker
mesh_terms: D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D005260:Female; D005922:Glomerulonephritis, IGA / Q000235:genetics / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D007678:Kidney Glomerulus / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D020537:RNA, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D035683:MicroRNAs; D020537:RNA, Small Nucleolar