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Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels

Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to thei...

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Autores principales: Jaeschke, Anna, Harvey, Nicholas R., Tsurkan, Mikhail, Werner, Carsten, Griffiths, Lyn R., Haupt, Larisa M., Bray, Laura J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169204/
https://www.ncbi.nlm.nih.gov/pubmed/34062095
http://dx.doi.org/10.1098/rsob.200388
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author Jaeschke, Anna
Harvey, Nicholas R.
Tsurkan, Mikhail
Werner, Carsten
Griffiths, Lyn R.
Haupt, Larisa M.
Bray, Laura J.
author_facet Jaeschke, Anna
Harvey, Nicholas R.
Tsurkan, Mikhail
Werner, Carsten
Griffiths, Lyn R.
Haupt, Larisa M.
Bray, Laura J.
author_sort Jaeschke, Anna
collection PubMed
description Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.
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spelling pubmed-81692042021-06-05 Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels Jaeschke, Anna Harvey, Nicholas R. Tsurkan, Mikhail Werner, Carsten Griffiths, Lyn R. Haupt, Larisa M. Bray, Laura J. Open Biol Methods and Techniques Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses. The Royal Society 2021-06-02 /pmc/articles/PMC8169204/ /pubmed/34062095 http://dx.doi.org/10.1098/rsob.200388 Text en © 2021 The Authors. https://creativecommons.org/licenses/by/4.0/Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, provided the original author and source are credited.
spellingShingle Methods and Techniques
Jaeschke, Anna
Harvey, Nicholas R.
Tsurkan, Mikhail
Werner, Carsten
Griffiths, Lyn R.
Haupt, Larisa M.
Bray, Laura J.
Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels
title Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels
title_full Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels
title_fullStr Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels
title_full_unstemmed Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels
title_short Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels
title_sort techniques for rna extraction from cells cultured in starpeg–heparin hydrogels
topic Methods and Techniques
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169204/
https://www.ncbi.nlm.nih.gov/pubmed/34062095
http://dx.doi.org/10.1098/rsob.200388
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