title: A novel in vitro transcription method for producing siRNAs without specific sequence requirements.
abstract: RNA interference is a powerful genetic approach for silencing of target genes in which 21-23 nucleotide small interfering RNAs (siRNAs) work as sequence-specific RNA interference (RNAi) mediators. Production of siRNAs by in vitro transcription is a useful method that it is simple, effective, and inexpensive. Commonly used in vitro transcription promoters use guanine as the transcription start nucleotide. This method is, therefore, restricted to generating siRNAs with target sequences of 5'-NNGN17C. Here we report a modified in vitro transcription method, in which pre-siRNAs containing the 5' overhanging single-stranded leader sequence are first synthesized, then a DNA oligonucleotide complementary to the leader sequence is added to form a RNA-DNA hybrid, which is removed using RNase H to obtain desired siRNAs. Using siRNAs prepared with this method, we successfully inhibited the expression of both exogenous and endogenous genes.
authors.affiliation: Laboratory of Protein Engineering, Beijing Institute of Biotechnology, 20 Fengtai Dongdajie Street, Beijing, 100071, China. xd_zhu@263.net
authors.forename: Xudong
authors.initials: X
authors.lastname: Zhu
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Ying
authors.initials: Y
authors.lastname: Dang
authors.forename: Yi
authors.initials: Y
authors.lastname: Feng
authors.forename: Peitang
authors.initials: P
authors.lastname: Huang
mesh_terms: D001483:Base Sequence; D020868:Gene Silencing; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins