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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3384639?pdf=render |
| _version_ | 1819044595066994688 |
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| author | Zhao Wang Huang Huang Hanshuo Zhang Changhong Sun Yang Hao Junyu Yang Yu Fan Jianzhong Jeff Xi |
| author_facet | Zhao Wang Huang Huang Hanshuo Zhang Changhong Sun Yang Hao Junyu Yang Yu Fan Jianzhong Jeff Xi |
| author_sort | Zhao Wang |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-21T10:15:10Z |
| format | Article |
| id | doaj.art-67874cac29294c01a5a238a8656db948 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T10:15:10Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-67874cac29294c01a5a238a8656db9482022-12-21T19:07:36ZengPublic 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 |
| 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 |
| title | 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_short | 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 |
| url | http://europepmc.org/articles/PMC3384639?pdf=render |
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