New methods for reverse transfection with siRNA from a solid surface.
We describe two efficient and inexpensive methods for reverse transfection with siRNA from a solid surface. One method involves localized reverse transfection from spots on a glass slide, which is mainly useful for making "transfection microarrays" (TMAs). The other involves reverse transfection in multiple wells of microtiter plates. Conditions for cell culture, preparation of reagents, and details of reverse transfection have been determined for several lines of cells, but we focus here on experiments with HeLa cells. In particular, we evaluated the efficiency of transfection, the cytotoxic effects of reverse transfection, and the efficiency of gene "knockdown" by transfection. We also performed phenotypic screening for a functional gene, during which cell viability was evaluated in terms of fluorescence from Calcein-AM. Our methods for reverse transfection with siRNA should be powerful tools that are useful for high-throughput analysis of functional genes.
Fujita
Satoshi
S
Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.
Takano
Kota
K
Ota
Eiji
E
Sano
Takuma
T
Yoshikawa
Tomohiro
T
Miyake
Masato
M
Miyake
Jun
J
D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering