title: RNA extraction for RNA sequencing of archival renal tissues.
abstract: BACKGROUND
  Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS.
  
  
  METHODS
  We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq(®) RNA Access Library Preparation Kit.
  
  
  CONCLUSIONS
  Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.
authors.affiliation: a Department of Clinical Medicine , University of Bergen ;
authors.forename: Lea
authors.initials: L
authors.lastname: Landolt
authors.affiliation: a Department of Clinical Medicine , University of Bergen ;
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Marti
authors.affiliation: a Department of Clinical Medicine , University of Bergen ;|b Department of Urology , Haukeland University Hospital , Bergen ;
authors.forename: Christian
authors.initials: C
authors.lastname: Beisland
authors.affiliation: c Department of Cancer Research and Molecular Medicine , Norwegian University of Science and Technology , Trondheim , Norway.
authors.forename: Arnar
authors.initials: A
authors.lastname: Flatberg
authors.affiliation: a Department of Clinical Medicine , University of Bergen ;
authors.forename: Oystein Solberg
authors.initials: OS
authors.lastname: Eikrem
mesh_terms: D000818:Animals; D001706:Biopsy; D019075:Electrophoresis, Capillary; D005260:Female; D005453:Fluorescence; D006801:Humans; D007668:Kidney / Q000737:chemistry*; D008297:Male; D012313:RNA / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017423:Sequence Analysis, RNA; D013056:Spectrophotometry, Ultraviolet
chemical_list: D012313:RNA
keywords: Formalin-fixed and paraffin embedded; RNA extraction; kidney biopsies; laser capture microdissection; next generation sequencing