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

Abstract

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 to 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 non-biased and high through-put 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 (IPF). 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.

Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution

“…Furthermore, maintenance of the cellular composition enables the use of cold stored PCLS for metabolomic, proteomic, or transcriptomic approaches (51,56).…”

An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling in ex vivo human precision-cut lung slices

“…However, to establish this hypothesis further, complementary analyses such as single cell RNA sequencing could be used. Importantly, these experiments are now facilitated due to recent advances in single cell and high quality RNA preparation from hPCLS 57 . Furthermore, to visualise the dynamics of single cell types in hPCLSs and test if they are consistent with our proteomics data, our live imaging set-up can be coupled to that of Akram et al 20 .…”

“…We propose that the complementary approach described here, using label-free imaging of hPCLS together with the measurement of soluble markers such as PRO-C1, FBN-C, C3M, could enable the characterisation of the dynamics of collagen synthesis and degradation (the fractional synthesis) ex vivo upon fibrosis progression. Furthermore, to dissect the role of single cell types in mediating the effects of various pro-fibrotic stimuli, our workflow could be coupled to the newly established single cell analysis pipeline in hPCLS by Stegmayr et al 57 . Also, as SHG does not require sample fixation, as it is the case for immunochemistry, this label-free approach should allow time-lapse studies monitoring the kinetics of ECM deposition over extended periods of time.…”

Precision-Cut Tumor Slices (PCTS) as an Ex Vivo Model in Immunotherapy Research

“…Although PCTS cannot be used directly for multi-omics analyses, they can still be dissociated or PCTS lysates can be generated and can subsequently be used for transcriptomic, proteomic and metabolomic assays [ 54 , 55 , 56 ]. A recent study using human lung PCTS followed a novel approach for PCTS RNA isolation, which involved agarose separation (PCTS are embedded in low-melting agarose, which can affect RNA quality) [ 56 ]. This approach led to RNA isolation of high yield and quality, which was successfully used in qRT-PCR and RNA sequencing studies [ 54 , 56 ].…”

“…A recent study using human lung PCTS followed a novel approach for PCTS RNA isolation, which involved agarose separation (PCTS are embedded in low-melting agarose, which can affect RNA quality) [ 56 ]. This approach led to RNA isolation of high yield and quality, which was successfully used in qRT-PCR and RNA sequencing studies [ 54 , 56 ]. Bulk RNA sequencing data from this approach were successfully used in deconvolution computational pipelines that led to the detection of various cell populations (including immune cell populations) within the human lung PCTS [ 54 , 56 ].…”