Determination of silencing potency of synthetic and RNase III-generated siRNA using a secreted luciferase assay.
RNA interference (RNAi) is an established tool for functional genomics studies that is also showing great potential for medical applications. Currently, one of the main goals in RNAi technology is the design and discovery of potent small interfering RNAs (siRNAs). Using a secreted luciferase from Gaussia princeps (GLuc), we developed a reporter assay, which allows for rapid potency assessment of siRNAs, by measuring luminescence activity in cell culture supernatants. The method was applied in microtiter plate format and validated by comparison to quantitative reverse transcription PCR (RT-PCR) and Western blot analysis. This reporter assay was used to evaluate in HeLa cells the potency of different siRNA mixtures generated by RNase III, or several synthetic siRNAs, all directed against human p53. The results show that all four siRNA mixtures generated by RNase III induce 50%-75% decrease of the reporter activity at less than 10 nM transfected concentration. In contrast, only one out of the five commercially available synthetic siRNAs showed comparable potency. These results suggest that one advantage of using enzymatic complex siRNA mixtures for RNAi is that, unlike single synthetic siRNAs, selecting a target region is not important to ensure potency.
Morlighem
Jean-Etienne
JE
New England Biolabs, Ipswich, MA 01938, USA.
Petit
Céline
C
Tzertzinis
George
G
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019556:COS Cells; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043244:Ribonuclease III / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D008156:Luciferases; D043244:Ribonuclease III