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.
Key wordsReverse transfection Solid surface Transfection with siRNA Transfection microarray (TMA) High-throughput phenotypic screening
This study was performed as part of “The Project for Development of Analytic Technology for Gene Functions with Cell Arrays”, which was funded by the New Energy and Industrial Technology Development Organization (NEDO) of Japan.