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Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response
Dendritic cells (DCs) transfected with total amplified tumor cell RNA have the potential to induce broad antitumor immune responses. However, analytical methods required for quantitatively assessing the integrity, fidelity, and functionality of the amplified RNA are lacking. We have developed a seri...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817939/ https://www.ncbi.nlm.nih.gov/pubmed/23653155 http://dx.doi.org/10.1038/mtna.2013.18 |
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author | Slagter-Jäger, Jacoba G Raney, Alexa Lewis, Whitney E DeBenedette, Mark A Nicolette, Charles A Tcherepanova, Irina Y |
author_facet | Slagter-Jäger, Jacoba G Raney, Alexa Lewis, Whitney E DeBenedette, Mark A Nicolette, Charles A Tcherepanova, Irina Y |
author_sort | Slagter-Jäger, Jacoba G |
collection | PubMed |
description | Dendritic cells (DCs) transfected with total amplified tumor cell RNA have the potential to induce broad antitumor immune responses. However, analytical methods required for quantitatively assessing the integrity, fidelity, and functionality of the amplified RNA are lacking. We have developed a series of assays including gel electrophoresis, northern blot, capping efficiency, and microarray analysis to determine integrity and fidelity and a model system to assess functionality after transfection into human DCs. We employed these tools to demonstrate that modifications to our previously reported total cellular RNA amplification process including the use of the Fast Start High Fidelity (FSHF) PCR enzyme, T7 Powerswitch primer, post-transcriptional capping and incorporation of a type 1 cap result in amplification of longer transcripts, greater translational competence, and a higher fidelity representation of the starting total RNA population. To study the properties of amplified RNA after transfection into human DCs, we measured protein expression levels of defined antigens coamplified with the starting total RNA populations and measured antigen-specific T cell expansion in autologous DC-T cell co-cultured in vitro. We conclude from these analyses that the improved RNA amplification process results in superior protein expression levels and a greater capacity of the transfected DCs to induce multifunctional antigen-specific memory T cells. |
format | Online Article Text |
id | pubmed-4817939 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-48179392016-04-17 Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response Slagter-Jäger, Jacoba G Raney, Alexa Lewis, Whitney E DeBenedette, Mark A Nicolette, Charles A Tcherepanova, Irina Y Mol Ther Nucleic Acids Methods - Original Article Dendritic cells (DCs) transfected with total amplified tumor cell RNA have the potential to induce broad antitumor immune responses. However, analytical methods required for quantitatively assessing the integrity, fidelity, and functionality of the amplified RNA are lacking. We have developed a series of assays including gel electrophoresis, northern blot, capping efficiency, and microarray analysis to determine integrity and fidelity and a model system to assess functionality after transfection into human DCs. We employed these tools to demonstrate that modifications to our previously reported total cellular RNA amplification process including the use of the Fast Start High Fidelity (FSHF) PCR enzyme, T7 Powerswitch primer, post-transcriptional capping and incorporation of a type 1 cap result in amplification of longer transcripts, greater translational competence, and a higher fidelity representation of the starting total RNA population. To study the properties of amplified RNA after transfection into human DCs, we measured protein expression levels of defined antigens coamplified with the starting total RNA populations and measured antigen-specific T cell expansion in autologous DC-T cell co-cultured in vitro. We conclude from these analyses that the improved RNA amplification process results in superior protein expression levels and a greater capacity of the transfected DCs to induce multifunctional antigen-specific memory T cells. Nature Publishing Group 2013-05 2013-05-07 /pmc/articles/PMC4817939/ /pubmed/23653155 http://dx.doi.org/10.1038/mtna.2013.18 Text en Copyright © 2013 American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-nd/3.0/ Molecular Therapy-Nucleic Acids is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Methods - Original Article Slagter-Jäger, Jacoba G Raney, Alexa Lewis, Whitney E DeBenedette, Mark A Nicolette, Charles A Tcherepanova, Irina Y Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response |
title | Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response |
title_full | Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response |
title_fullStr | Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response |
title_full_unstemmed | Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response |
title_short | Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response |
title_sort | evaluation of rna amplification methods to improve dc immunotherapy antigen presentation and immune response |
topic | Methods - Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817939/ https://www.ncbi.nlm.nih.gov/pubmed/23653155 http://dx.doi.org/10.1038/mtna.2013.18 |
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