PMID- 28274266
OWN - NLM
STAT- MEDLINE
DCOM- 20170317
LR  - 20181113
IS  - 1756-0500 (Electronic)
IS  - 1756-0500 (Linking)
VI  - 10
IP  - 1
DP  - 2017 Mar 9
TI  - RNA isolation from precision-cut lung slices (PCLS) from different species.
PG  - 121
LID - 10.1186/s13104-017-2447-6 [doi]
LID - 121
AB  - 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.
FAU - Niehof, Monika
AU  - Niehof M
AUID- ORCID: 0000-0001-5687-3052
AD  - 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.
FAU - Hildebrandt, Tobias
AU  - Hildebrandt T
AD  - Immunology and Respiratory, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim 
      am Rhein, Germany.
FAU - Danov, Olga
AU  - Danov O
AD  - 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.
FAU - Arndt, Kirsten
AU  - Arndt K
AD  - Immunology and Respiratory, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim 
      am Rhein, Germany.
FAU - Koschmann, Jeannette
AU  - Koschmann J
AD  - geneXplain GmbH, 38302, Wolfenbuttel, Germany.
FAU - Dahlmann, Franziska
AU  - Dahlmann F
AD  - 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.
AD  - Pathology Unit, German Primate Center GmbH, Leibniz-Institute for Primate 
      Research, Kellnerweg 4, 37077, Gottingen, Germany.
FAU - Hansen, Tanja
AU  - Hansen T
AD  - 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.
FAU - Sewald, Katherina
AU  - Sewald K
AD  - 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.
LA  - eng
PT  - Journal Article
DEP - 20170309
PL  - England
TA  - BMC Res Notes
JT  - BMC research notes
JID - 101462768
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Aged
MH  - Animals
MH  - Callithrix
MH  - Female
MH  - Humans
MH  - Lung/*chemistry/surgery
MH  - Macaca mulatta
MH  - Magnets
MH  - Male
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - Microarray Analysis
MH  - Microtomy/instrumentation/*methods
MH  - Middle Aged
MH  - Oligonucleotide Array Sequence Analysis/*methods/statistics & numerical data
MH  - RNA/genetics/*isolation & purification
MH  - Rats
MH  - Rats, Wistar
MH  - Reverse Transcriptase Polymerase Chain Reaction
MH  - *Transcriptome
PMC - PMC5343379
OTO - NOTNLM
OT  - Ex vivo
OT  - Lung material
OT  - Lung tissue
OT  - Microarray
OT  - Organotypic tissue
OT  - RNA extraction
OT  - RNA quality
OT  - RTqPCR
EDAT- 2017/03/10 06:00
MHDA- 2017/03/18 06:00
PMCR- 2017/03/09
CRDT- 2017/03/10 06:00
PHST- 2016/08/02 00:00 [received]
PHST- 2017/03/01 00:00 [accepted]
PHST- 2017/03/10 06:00 [entrez]
PHST- 2017/03/10 06:00 [pubmed]
PHST- 2017/03/18 06:00 [medline]
PHST- 2017/03/09 00:00 [pmc-release]
AID - 10.1186/s13104-017-2447-6 [pii]
AID - 2447 [pii]
AID - 10.1186/s13104-017-2447-6 [doi]
PST - epublish
SO  - BMC Res Notes. 2017 Mar 9;10(1):121. doi: 10.1186/s13104-017-2447-6.