A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification.
RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3' end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed 'crook' siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics.
Jiang
Ming
M
YCR P53 Research Laboratory, Department of Biology, University of York, York YO10 5DD, UK.
Arzumanov
Andrey A
AA
Gait
Michael J
MJ
Milner
Jo
J
D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000032:analysis* / Q000737:chemistry*; D013698:Templates, Genetic; D014162:Transfection
D017931:DNA Primers; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C059731:oncogene protein E7, Human papillomavirus type 16