A novel in vitro transcription method for producing siRNAs without specific sequence requirements.
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.
Zhu
Xudong
X
Laboratory of Protein Engineering, Beijing Institute of Biotechnology, 20 Fengtai Dongdajie Street, Beijing, 100071, China. xd_zhu@263.net
Li
Tao
T
Dang
Ying
Y
Feng
Yi
Y
Huang
Peitang
P
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
D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins