An efficient and fully automated high-throughput transfection method for genome-scale siRNA screens.
J Biomol Screen
; 13(2): 142-8, 2008 Feb.
Article
em En
| MEDLINE
| ID: mdl-18216392
ABSTRACT
RNA interference (RNAi), combined with the availability of genome sequences, provides an unprecedented opportunity for the massive and parallel investigations of gene function. Small interfering RNA (siRNA) represents a popular and quick approach of RNAi for in vitro loss-of-function genetic screens. Efficient transfection of siRNA is critical for unambiguous interpretation of screen results and thus overall success of any siRNA screen. A high-throughput, lipid-based transfection method for siRNA was developed that can process eighty 384-well microplates in triplicate (for a total of 30,720 unique transfections) in 8 h. Transfection throughput was limited only by the speed of robotics, whereas the cost of screening was reduced. As a proof of principle, a genome-scale screen with a library of 22,108 siRNAs was performed to identify the genes sensitizing cells to mitomycin C at concentrations of 0, 20, and 60 nM. Transfection efficiency, performances of control siRNAs, and other quality metrics were monitored and demonstrated that the new, optimized transfection protocol produced high-quality results throughout the screen.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Transfecção
/
Genoma Humano
/
RNA Interferente Pequeno
Tipo de estudo:
Evaluation_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article