Nucleic Acids Research Advance Access originally published online on October 31, 2008
Nucleic Acids Research 2008 36(22):e149; doi:10.1093/nar/gkn715
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Nucleic Acids Research, 2008, Vol. 36, No. 22 e149
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Rolling circle amplification-mediated hairpin RNA (RMHR) library construction in plants
1Biotechnology Research Institute/The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China and 2CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia
*To whom correspondence should be addressed. Fax: +86 10 62133870; Email: wanglei{at}caas.net.cn
Received July 22, 2008. Revised September 24, 2008. Accepted September 30, 2008.
Long hairpin RNA (lhRNA) construct-induced gene silencing facilitates the study of gene function in plants and animals, but constructing multiple lhRNA vectors using traditional approaches is both time-consuming and costly. Also, most of the existing approaches are based on sequence-specific cloning of individual sequences, and are therefore not suitable for preparing hpRNA libraries from a pool of mixed target sequences. Here we describe a rolling-circle amplification (RCA)-mediated hpRNA (RMHR) construction system suitable for generating libraries of lhRNA constructs from any gene of interest or pool of genes. Using RMHR we successfully generated a lhRNA library from a Arabidopsis cDNA population containing known and unknown genes, with an average size of 500–800 bp for the inverted-repeat inserts. To validate the RMHR system, lhRNA constructs targeting the β-glucuronidase (GUS) gene were tested using Agrobacterium infiltration and shown to be effective at inducing GUS silencing in tobacco leaves. Our results indicate that the RMHR technique permits rapid, efficient and low-cost preparation of genome-wide lhRNA expression libraries.