A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.
The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect...
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doaj-67874cac29294c01a5a238a8656db9482020-11-25T01:29:12ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0176e3941910.1371/journal.pone.0039419A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.Zhao WangHuang HuangHanshuo ZhangChanghong SunYang HaoJunyu YangYu FanJianzhong Jeff XiThe chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.http://europepmc.org/articles/PMC3384639?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Zhao Wang Huang Huang Hanshuo Zhang Changhong Sun Yang Hao Junyu Yang Yu Fan Jianzhong Jeff Xi |
spellingShingle |
Zhao Wang Huang Huang Hanshuo Zhang Changhong Sun Yang Hao Junyu Yang Yu Fan Jianzhong Jeff Xi A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs. PLoS ONE |
author_facet |
Zhao Wang Huang Huang Hanshuo Zhang Changhong Sun Yang Hao Junyu Yang Yu Fan Jianzhong Jeff Xi |
author_sort |
Zhao Wang |
title |
A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs. |
title_short |
A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs. |
title_full |
A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs. |
title_fullStr |
A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs. |
title_full_unstemmed |
A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs. |
title_sort |
magnetic bead-integrated chip for the large scale manufacture of normalized esirnas. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2012-01-01 |
description |
The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications. |
url |
http://europepmc.org/articles/PMC3384639?pdf=render |
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