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

Stegmayr, John LU ; Alsafadi, Hani N LU ; Langwinski, Wojciech LU ; Niroomand, Anna LU ; Lindstedt, Sandra LU ; Leigh, Nicholas D LU and Wagner, Darcy E LU (2021) In American Journal of Physiology: Lung Cellular and Molecular Physiology 320(2). p.232-240
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... (More)

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.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
American Journal of Physiology: Lung Cellular and Molecular Physiology
volume
320
issue
2
pages
232 - 240
publisher
American Physiological Society
external identifiers
  • pmid:33112185
ISSN
1522-1504
DOI
10.1152/ajplung.00401.2020
language
English
LU publication?
yes
id
7722000c-549f-4a1d-8a0c-b2eaae469303
date added to LUP
2020-11-05 10:12:20
date last changed
2021-04-21 07:34:16
@article{7722000c-549f-4a1d-8a0c-b2eaae469303,
  abstract     = {<p>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.</p>},
  author       = {Stegmayr, John and Alsafadi, Hani N and Langwinski, Wojciech and Niroomand, Anna and Lindstedt, Sandra and Leigh, Nicholas D and Wagner, Darcy E},
  issn         = {1522-1504},
  language     = {eng},
  number       = {2},
  pages        = {232--240},
  publisher    = {American Physiological Society},
  series       = {American Journal of Physiology: Lung Cellular and Molecular Physiology},
  title        = {Isolation of high yield and quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts},
  url          = {http://dx.doi.org/10.1152/ajplung.00401.2020},
  doi          = {10.1152/ajplung.00401.2020},
  volume       = {320},
  year         = {2021},
}