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Generation of shrnas from randomized oligonucleotides

Abstract

Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries from either individually made gene specific sequences or cDNA libraries, there is no report on constructing randomized shRNA libraries, which could provide a good alternative to these existing libraries. We have developed a method of constructing shRNAs from randomized oligonucleotides. Through this method, one can generate a partially or fully randomized shRNA library for various functional analyses. We validated this procedure by constructing a p53-specific shRNA. Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells. We then made a partially randomized shRNA library. Sequencing of 15 randomly picked cloned confirmed the randomness of the library. Therefore, the library can be used for various functional assays, such as target validation when a suitable screening or selection method is available.

Abbreviations

bp:

base pair

RNAi:

RNA interference

shRNA:

short hairpin RNA

siRNA:

short interfering RNA

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Correspondence to Yin-Yuan Mo.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Wu, H., Dinh, A. & Mo, YY. Generation of shrnas from randomized oligonucleotides. Biol. Proced. Online 9, 9–17 (2007). https://doi.org/10.1251/bpo129

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  • DOI: https://doi.org/10.1251/bpo129

Indexing terms

  • RNA Interference
  • Small Interfering, RNA
  • Oligonucleotides