title: A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.
abstract: 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.
authors: Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China.
authors: Zhao
authors: Z
authors: Wang
authors: Huang
authors: H
authors: Huang
authors: Hanshuo
authors: H
authors: Zhang
authors: Changhong
authors: C
authors: Sun
authors: Yang
authors: Y
authors: Hao
authors: Junyu
authors: J
authors: Yang
authors: Yu
authors: Y
authors: Fan
authors: Jianzhong Jeff
authors: JJ
authors: Xi
mesh_terms: D004722:Endoribonucleases / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D034741:RNA, Small Interfering / Q000138:chemical synthesis* / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering; D004722:Endoribonucleases