ScienceAsia 33 (2007): 035-039 |doi: 10.2306/scienceasia1513-1874.2007.33.035

A Simple and Cost Effective Method to Generate dsRNA for RNAi Studies in Invertebrates

Chalermporn Ongvarrasopone^a*, Yaowaluck Roshorm^a and Sakol Panyim^a,b

 
ABSTRACT: Using an E. coli strain deficient in ribonuclease III activity, we have developed a simple and cost effective in vivo bacterial expression system to generate large amounts of double-stranded RNA (dsRNA). This method, which involves E. coli culture, RNase A treatment of lysed cells and total RNA extraction, is easier and less expensive than traditional in vitro transcription techniques. The system was validated by knocking down yellow head virus replication in shrimp OKA cell cultures. Results showed that the dsRNAs prepared in vitro and in vivo possessed similar potency in inhibiting viral replication. This methodology gives an alternative means to prepare large amounts of dsRNA at low cost.

KEYWORDS: double-stranded RNA, E. coli expression, in vitro transcription, yellow head virus, shrimp.

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^a Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Nakhon Pathom 73170,
Thailand.
^b Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400,
Thailand.

* Corresponding author, E-mail: mbcov@mahidol.ac.th

Received 21 Jun 2005, Accepted 4 Aug 2006