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BMC Res Notes. 2017 Mar 9;10(1):121. doi: 10.1186/s13104-017-2447-6.

RNA isolation from precision-cut lung slices (PCLS) from different species.

Author information

1
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.
2
Immunology and Respiratory, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany.
3
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.
4
geneXplain GmbH, 38302, Wolfenbüttel, Germany.
5
Pathology Unit, German Primate Center GmbH, Leibniz-Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany.

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.

KEYWORDS:

Ex vivo; Lung material; Lung tissue; Microarray; Organotypic tissue; RNA extraction; RNA quality; RTqPCR

PMID:
28274266
PMCID:
PMC5343379
DOI:
10.1186/s13104-017-2447-6
[Indexed for MEDLINE]
Free PMC Article

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