Quantitative evaluation of siRNA delivery in vivo.
RNA
; 16(12): 2553-63, 2010 Dec.
Article
in En
| MEDLINE
| ID: mdl-20940339
ABSTRACT
Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.
Full text:
1
Database:
MEDLINE
Main subject:
Gene Transfer Techniques
/
RNA, Small Interfering
Type of study:
Diagnostic_studies
/
Evaluation_studies
Limits:
Animals
/
Female
/
Humans
Language:
En
Journal:
RNA
Journal subject:
BIOLOGIA MOLECULAR
Year:
2010
Type:
Article
Affiliation country:
United States