Techniques for RNA extraction from cells cultured in starPEG-heparin hydrogels.

Mikhail Tsurkan,Nicholas R Harvey,Lyn R Griffiths,Laura J Bray,Anna Jaeschke,Carsten Werner,Larisa M Haupt

doi:10.1098/rsob.200388

Abstract

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.

Highlights

Relevant in vitro models considering the cellular, biochemical and biomechanical aspects of the tissue microenvironment are essential to study the molecular mechanism of diseases such as cancer [1,2]
We evaluated RNA yield and quality as well as the performance of cDNA synthesized from the various RNA preparations in real-time quantitative polymerase chain reaction (qPCR) (RT-qPCR)
For the isolation of RNA from cells cultured in starPEG– heparin hydrogels, two different isolation kits were tested which use specific techniques for cell lysis

Summary

Introduction

Relevant in vitro models considering the cellular, biochemical and biomechanical aspects of the tissue microenvironment are essential to study the molecular mechanism of diseases such as cancer [1,2]. Integrated RGD (arginine–glycine–aspartate) motifs provide cell binding sites, and matrix metalloproteinase (MMP)-responsive sequences allow for cells to locally remodel the microenvironment. Signalling cues, such as growth factors, can be embedded using the negative charge of the heparin molecules [5]. StarPEG–heparin hydrogels have been used for a variety of research applications such as Alzheimer’s disease modelling [6], cartilage regeneration [7] and in vitro cancer modelling [8,9,10] These hydrogels are ideal models for tumour angiogenesis studies as their nature allows for vascular cells to extend, interconnect and form networks in vitro [11,12,13]

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Results

Conclusion