A modified protocol for successful miRNA profiling in human precision-cut lung slices (PCLS)

BMC Research Notes

Stella Marie Reamon-Buettner¹  Tanja Hansen¹  Monika Niehof¹  Olga Danov¹  Katherina Sewald¹ 

[1] Department of Preclinical Pharmacology and In Vitro Toxicology, Fraunhofer Institute for Toxicology and Experimental Medicine, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany;

关键词: miRNA extraction;    RTqPCR;    Microarray;    miRNA array;    RNA quality;    Lung tissue;    Lung material;    Ex vivo;    PCLS;    Organotypic tissue;   

DOI  :  10.1186/s13104-021-05674-w

来源: Springer

【 摘 要 】

ObjectiveHuman precision cut lung slices (PCLS) are widely used as an ex vivo model system for drug discovery and development of new therapies. PCLS reflect the functional heterogeneity of lung tissue and possess relevant lung cell types. We thus determined the use of PCLS in studying non-coding RNAs notably miRNAs, which are important gene regulatory molecules. Since miRNAs play key role as mediators of respiratory diseases, they can serve as valuable prognostic or diagnostic biomarkers, and in therapeutic interventions, of lung diseases. A technical limitation though is the vast amount of agarose in PCLS which impedes (mi)RNA extraction by standard procedures. Here we modified our recently published protocol for RNA isolation from PCLS to enable miRNA readouts.ResultsThe modified method relies on the separation of lysis and precipitation steps, and a clean-up procedure with specific magnetic beads. We obtained successfully quality miRNA amenable for downstream applications such as RTqPCR and whole transcriptome miRNA analysis. Comparison of miRNA profiles in PCLS with published data from human lung, identified all important miRNAs regulated in IPF, COPD, asthma or lung cancer. Therefore, this shows suitability of the method for analyzing miRNA targets and biomarkers in the valuable human PCLS model.

【 授权许可】

CC BY