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New Methods for Reverse Transfection with siRNA from a Solid Surface

  • Satoshi Fujita
  • Kota Takano
  • Eiji Ota
  • Takuma Sano
  • Tomohiro Yoshikawa
  • Masato Miyake
  • Jun Miyake
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 623)

Abstract

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 words

Reverse transfection Solid surface Transfection with siRNA Transfection microarray (TMA) High-throughput phenotypic screening 

Notes

Acknowledgments

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.

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Copyright information

©  Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Satoshi Fujita
    • 1
  • Kota Takano
    • 1
  • Eiji Ota
    • 1
  • Takuma Sano
    • 1
  • Tomohiro Yoshikawa
    • 1
  • Masato Miyake
    • 1
  • Jun Miyake
    • 1
    • 2
  1. 1.Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST)TokyoJapan
  2. 2.Department of Bioengineering, School of EngineeringUniversity of TokyoTokyoJapan

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