title: RNA isolation from precision-cut lung slices (PCLS) from different species.
abstract: BACKGROUND
  Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. To enable wider implementation in research and development, these new biologically relevant organ models are being constantly refined. A very important issue is to improve the preparation of high-quality RNA (ribonucleic acid) from PCLS for drug discovery and development of new therapies. Gene expression analysis at different levels is used as an important experimental readout. Genome-wide analysis using microarrays is mostly applied for biomarker selection in disease models or in comprehensive toxicological studies. Specific biomarker testing by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is often used in efficacy studies. Both applications require high-quality RNA as starting material for the generation of reliable data. Additionally, a small number of slices should be sufficient for satisfactory RNA isolation to allow as many experimental conditions as possible to be covered with a given tissue sample. Unfortunately, the vast amount of agarose in PCLS impedes RNA extraction according to the standard procedures.
  
  
  RESULTS
  We established an optimized protocol for RNA isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques based on the separation of lysis and precipitation steps and a magnetic-bead cleanup procedure. The resulting RNA is of high purity and possesses a high degree of integrity. There are no contaminations affecting RTqPCR efficiency or any enzymatic step in sample preparation for microarray analysis.
  
  
  CONCLUSIONS
  In summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications.
authors.affiliation: Division of Preclinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Excellence Cluster REBIRTH, Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany. monika.niehof@item.fraunhofer.de.
authors.forename: Monika
authors.identifier: http://orcid.org/0000-0001-5687-3052
authors.initials: M
authors.lastname: Niehof
authors.affiliation: Immunology and Respiratory, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Hildebrandt
authors.affiliation: Division of Preclinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Excellence Cluster REBIRTH, Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Danov
authors.affiliation: Immunology and Respiratory, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Arndt
authors.affiliation: geneXplain GmbH, 38302, Wolfenbüttel, Germany.
authors.forename: Jeannette
authors.initials: J
authors.lastname: Koschmann
authors.affiliation: Division of Preclinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Excellence Cluster REBIRTH, Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany.|Pathology Unit, German Primate Center GmbH, Leibniz-Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Dahlmann
authors.affiliation: Division of Preclinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Excellence Cluster REBIRTH, Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Hansen
authors.affiliation: Division of Preclinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Excellence Cluster REBIRTH, Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany.
authors.forename: Katherina
authors.initials: K
authors.lastname: Sewald
mesh_terms: D000368:Aged; D000818:Animals; D002144:Callithrix; D005260:Female; D006801:Humans; D008168:Lung / Q000737:chemistry* / Q000601:surgery; D008253:Macaca mulatta; D059346:Magnets; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D046228:Microarray Analysis; D008867:Microtomy / Q000295:instrumentation / Q000379:methods*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000706:statistics & numerical data; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059467:Transcriptome
chemical_list: D012313:RNA
keywords: Ex vivo; Lung material; Lung tissue; Microarray; Organotypic tissue; RNA extraction; RNA quality; RTqPCR