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Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines

The use of RNA interference (RNAi) to deplete individual proteins from cells or tissue has revolutionised our ability to characterise gene function. The placenta is an attractive target for studies in which the role of specific proteins can be compared with cell culture models and explanted villous...

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Detalles Bibliográficos
Autores principales: Forbes, K., Desforges, M., Garside, R., Aplin, J.D., Westwood, M.
Formato: Texto
Lenguaje:English
Publicado: W.B. Saunders 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634981/
https://www.ncbi.nlm.nih.gov/pubmed/19012963
http://dx.doi.org/10.1016/j.placenta.2008.10.003
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author Forbes, K.
Desforges, M.
Garside, R.
Aplin, J.D.
Westwood, M.
author_facet Forbes, K.
Desforges, M.
Garside, R.
Aplin, J.D.
Westwood, M.
author_sort Forbes, K.
collection PubMed
description The use of RNA interference (RNAi) to deplete individual proteins from cells or tissue has revolutionised our ability to characterise gene function. The placenta is an attractive target for studies in which the role of specific proteins can be compared with cell culture models and explanted villous tissue where physiological function can be maintained ex vivo. In this study, we compared a variety of commercially available reagents and approaches to define methods for efficient delivery of siRNA to placental cells. Protocols optimised using fluorescently-labelled siRNA were subsequently tested using siRNA sequences that target placental alkaline phosphatase (PLAP), chosen because of its high abundance in trophoblast. mRNA abundance was assayed using qRT-PCR, and the effect on protein was examined using immunolocalisation. We report that different protocols are required for BeWo choriocarcinoma cells (nucleofection), primary cytotrophoblast cells (lipid-based transfection) and villous tissue explants (nucleofection). The results provide guidelines for optimal siRNA-mediated knockdown in these three models of the human placenta.
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spelling pubmed-26349812009-02-12 Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines Forbes, K. Desforges, M. Garside, R. Aplin, J.D. Westwood, M. Placenta Technical Note The use of RNA interference (RNAi) to deplete individual proteins from cells or tissue has revolutionised our ability to characterise gene function. The placenta is an attractive target for studies in which the role of specific proteins can be compared with cell culture models and explanted villous tissue where physiological function can be maintained ex vivo. In this study, we compared a variety of commercially available reagents and approaches to define methods for efficient delivery of siRNA to placental cells. Protocols optimised using fluorescently-labelled siRNA were subsequently tested using siRNA sequences that target placental alkaline phosphatase (PLAP), chosen because of its high abundance in trophoblast. mRNA abundance was assayed using qRT-PCR, and the effect on protein was examined using immunolocalisation. We report that different protocols are required for BeWo choriocarcinoma cells (nucleofection), primary cytotrophoblast cells (lipid-based transfection) and villous tissue explants (nucleofection). The results provide guidelines for optimal siRNA-mediated knockdown in these three models of the human placenta. W.B. Saunders 2009-02 /pmc/articles/PMC2634981/ /pubmed/19012963 http://dx.doi.org/10.1016/j.placenta.2008.10.003 Text en © 2009 Elsevier Ltd. https://creativecommons.org/licenses/by/3.0/ Open Access under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) license
spellingShingle Technical Note
Forbes, K.
Desforges, M.
Garside, R.
Aplin, J.D.
Westwood, M.
Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines
title Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines
title_full Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines
title_fullStr Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines
title_full_unstemmed Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines
title_short Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines
title_sort methods for sirna-mediated reduction of mrna and protein expression in human placental explants, isolated primary cells and cell lines
topic Technical Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634981/
https://www.ncbi.nlm.nih.gov/pubmed/19012963
http://dx.doi.org/10.1016/j.placenta.2008.10.003
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