Isolation of high-yield and -quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts.

Stegmayr J, Alsafadi HN, LangwiƄski W, Niroomand A, Lindstedt S, Leigh ND, Wagner DE

Am J Physiol Lung Cell Mol Physiol 320 (2) L232-L240 [2021-02-01; online 2020-10-28]

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared with animal models. Several experimental readouts have been established for use with PCLS, but obtaining high-yield and -quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for nonbiased and high-throughput analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high-quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis. We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.

Bioinformatics Support for Computational Resources [Service]

Clinical Genomics Lund [Service]

PubMed 33112185

DOI 10.1152/ajplung.00401.2020

Crossref 10.1152/ajplung.00401.2020

Publications 9.5.0