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

Monika Niehof,Franziska Dahlmann,Tobias Hildebrandt,Tanja Hansen,Jeannette Koschmann,Katherina Sewald,Kirsten Arndt,Olga Danov

doi:10.1186/s13104-017-2447-6

Abstract

BackgroundFunctional 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.ResultsWe 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.ConclusionsIn summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications.

Highlights

Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research
A very important issue is to improve the preparation of high-quality RNA from PCLS for drug discovery and development of new therapies
Our goal was to establish a protocol for RNA isolation from PCLS from different species that would yield RNA of high purity and integrity—even in the presence of a large amount of agarose in PCLS, which impedes RNA extraction—and is well suited for gene expression analysis as a downstream application

Summary

Introduction

Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. 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. 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. There have been significant improvements in the preparation and culture of ex vivo tissues, so that thin and precise sections are obtained, which can be maintained for days, weeks, and even months [1,2,3] Today they form the basis for high quality research for drug discovery and new therapy development.

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