RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Burgess, Kyle A.; Workman, Victoria L.; Elsawy, Mohamed A.; Miller, Aline F.; Oceandy, Delvac; Saiani, Alberto

doi:10.1371/journal.pone.0197517

Cited by 14 publications

(15 citation statements)

References 35 publications

(36 reference statements)

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“…2. This finding broadly supports the work of other studies [26][27][28] linking natural polymers with positively charged amine groups with decreases in RNA yield or quality. Several possible explanations could describe these findings.…”

Section: Discussionsupporting

confidence: 90%

pH-dependent RNA isolation from cells encapsulated in chitosan-based biomaterials

Farrag

Abri

Leipzig

2020

International Journal of Biological Macromolecules

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Chitosan has emerged as a useful biomaterial employed in tissue engineering and drug delivery applications due to its tunable and interesting properties. However, chitosan is protonated at biological pH and thus carries positive charges, which renders chitosan incompatible with conventional methods of RNA extraction. RNA extraction is an important step in investigating cell responses and behavior through studying their gene expression transcriptional profile. While some researchers have tried different techniques to improve the yield and purity of RNA extracted from cells encapsulated in chitosan-based biomaterials, no single study has investigated the effects of manipulating pH of the homogenate during RNA extraction on the yield and quality of total RNA. This study confirms the release and binding of RNA from chitosan to be pH dependent while analyzing the impact of pH changes during the tissue disruption and homogenization step of extraction on the resulting yield and quality of isolated RNA. This concept was applied to three commonly used methods of RNA extraction, using adult neural stem cells (aNSPCs) encapsulated within methacrylamide chitosan (MAC) as model chitosan-based bioscaffold. High pH conditions resulted in high yields with good quality using both TRIzol and CTAB. pH of the homogenate did not affect RNeasy spin columns, which works best in neutral conditions with good quality, however, the overall yield was low. Results in total show that pH affects RNA interaction with a chitosan-based bioscaffold, and thus alters the concentration, purity, and integrity of isolated RNA, dependent on the method used.

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Section: Discussionsupporting

confidence: 90%

pH-dependent RNA isolation from cells encapsulated in chitosan-based biomaterials

Farrag

Abri

Leipzig

2020

International Journal of Biological Macromolecules

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“…This reduction in the yield was probably due to the presence of fibrils. A study by Burgess et al, 2018 (9) undertook a comparative analysis of RNA extraction from cells encapsulated in self-assembling peptide hydrogels using two different extraction principles: solution-based extraction and direct solid-state binding of RNA and showed the latter was a more efficient method of extraction (9). In line with this study, we have shown that the use of a direct solid-state binding of RNA extraction method allowed the extraction of RNA of PeptiGel ® -organoids.…”

Section: Discussionmentioning

confidence: 99%

“…In line with this study, we have shown that the use of a direct solid-state binding of RNA extraction method allowed the extraction of RNA of PeptiGel ® -organoids. Burgess et al, also showed that the use of pronase, a broad-spectrum enzyme solution reduced the amount of fibril present and increased RNA yield (9). Therefore, future investigation will aim to increase the yield of the RNA content by employing the use and impact of pronase to digest the organoids lysate before being subjected to RNA analysis.…”

Section: Discussionmentioning

confidence: 99%

“…CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 1, 2021. ; https://doi.org/10.1101/2021.03.01.433333 doi: bioRxiv preprint 10 comparative analysis of RNA extraction from cells encapsulated in self-assembling peptide hydrogels using two different extraction principles: solution-based extraction and direct solid-state binding of RNA and showed the latter was a more efficient method of extraction (9). In line with this study, we have shown that the use of a direct solid-state binding of RNA extraction method allowed the extraction of RNA of PeptiGel®-organoids.…”

Section: Rna Extraction and Pcr Analysis Of Cytokeratin-18 Genes Frommentioning

confidence: 99%

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Self-Assembling Peptide Hydrogels - PeptiGels^®as a Platform for Hepatic Organoid Culture

Olayanju

Ansari

et al. 2021

Preprint

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A major challenge in advancing preclinical studies is the lack of robust in vitro culture systems that fully recapitulate the in vivo scenario together with limited clinical translational to humans. Organoids, as 3-dimensional (3D) self-replicating structures are increasingly being shown as powerful models for ex vivo experimentation in the field of regenerative medicine and drug discovery. Organoid formation requires the use of extracellular matrix (ECM) components to provide a 3D platform. However, the most commonly used ECM, essential for maintaining organoid growth is Matrigel and is derived from a tumorigenic source which limits its translational ability. PeptiGels® which are self-assembling peptide hydrogels present as alternatives to traditional ECM for use in 3D culture systems. Synthetic PeptiGels® are non-toxic, biocompatible, biodegradable and can be tuneable to simulate different tissue microenvironments. In this study, we validated the use of different types of PeptiGels® for porcine hepatic organoid growth. Hepatic organoids were assessed morphologically and using molecular techniques to determine the optimum PeptiGel® formulation. The outcome clearly demonstrated the ability of PeptiGel® to support organoid growth and offer themselves as a technological platform for 3D cultured physiologically and clinically relevant data.

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“…One way to avoid this is to use cells from different species, so that species-specific primers can be used for identifying transcript changes. Alternatively, cells can be extracted from the hydrogel and separated by flow cytometry, although enzymes used to digest the polymer network may degrade the cellular material to be measured or interfere in the subsequent detection [ 166 ]. Changes in junctional complex mRNA expression in brain endothelial cells generally correlate with the protein level changes and functional changes in TEER [ 167 ].…”

Section: Outcome Measurements Of Bbb Functionality In Nvu Modelsmentioning

confidence: 99%

3D hydrogel models of the neurovascular unit to investigate blood–brain barrier dysfunction

Potjewyd

Hooper

2021

Neuronal Signaling

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The neurovascular unit (NVU), consisting of neurons, glial cells, vascular cells (endothelial cells, pericytes and vascular smooth muscle cells (VSMCs)) together with the surrounding extracellular matrix (ECM), is an important interface between the peripheral blood and the brain parenchyma. Disruption of the NVU impacts on blood–brain barrier (BBB) regulation and underlies the development and pathology of multiple neurological disorders, including stroke and Alzheimer’s disease (AD). The ability to differentiate induced pluripotent stem cells (iPSCs) into the different cell types of the NVU and incorporate them into physical models provides a reverse engineering approach to generate human NVU models to study BBB function. To recapitulate the in vivo situation such NVU models must also incorporate the ECM to provide a 3D environment with appropriate mechanical and biochemical cues for the cells of the NVU. In this review, we provide an overview of the cells of the NVU and the surrounding ECM, before discussing the characteristics (stiffness, functionality and porosity) required of hydrogels to mimic the ECM when incorporated into in vitro NVU models. We summarise the approaches available to measure BBB functionality and present the techniques in use to develop robust and translatable models of the NVU, including transwell models, hydrogel models, 3D-bioprinting, microfluidic models and organoids. The incorporation of iPSCs either without or with disease-specific genetic mutations into these NVU models provides a platform in which to study normal and disease mechanisms, test BBB permeability to drugs, screen for new therapeutic targets and drugs or to design cell-based therapies.

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RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Cited by 14 publications

References 35 publications

pH-dependent RNA isolation from cells encapsulated in chitosan-based biomaterials

pH-dependent RNA isolation from cells encapsulated in chitosan-based biomaterials

Self-Assembling Peptide Hydrogels - PeptiGels^®as a Platform for Hepatic Organoid Culture

3D hydrogel models of the neurovascular unit to investigate blood–brain barrier dysfunction

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RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Cited by 14 publications

References 35 publications

pH-dependent RNA isolation from cells encapsulated in chitosan-based biomaterials

pH-dependent RNA isolation from cells encapsulated in chitosan-based biomaterials

Self-Assembling Peptide Hydrogels - PeptiGels®as a Platform for Hepatic Organoid Culture

3D hydrogel models of the neurovascular unit to investigate blood–brain barrier dysfunction

Contact Info

Product

Resources

About

Self-Assembling Peptide Hydrogels - PeptiGels^®as a Platform for Hepatic Organoid Culture