ABSTRACT
Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Appendix 4.1 Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces the posttranscriptional degradation of homologous transcripts. This phenomenon has been observed in a variety of organisms, including plants, fungi, insects, protozoans, and mammals.1 Introduction of dsRNA into cells leads to the sequence-specific destruction of endogenous RNAs that are complementary to the dsRNA.2 According to the prevailing model, long dsRNA molecules are cleaved into short interfering dsRNA species (siRNAs) of about 21 to 24 nt.3 In mammalian cells, long dsRNA (more than 30 nt) molecules cannot be used since they elicit a nonspecific interferon response.4 This problem can be avoided with siRNAs of 21 nt. These siRNAs efficiently inhibit target gene expression in a sequence-specific manner5 but are too short (< 30 bp) to trigger the nonspecific dsRNA responses. Since RNAi technology has begun to be applied to mammalian cells, many researchers have performed it with expensive chemically synthesized siRNAs. However, we have recently reported6 a technically simple and inexpensive alternative method for the production of siRNAs. It makes use of in vitro transcription with T7 RNA polymerase from DNA oligonucleotides. This chapter provides a more comprehensive description of this method with detailed experimental protocols.
