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Reverse transfection on cell arrays for high content screening microscopy.

Holger Erfle1, Beate Neumann, Urban Liebel

  • 1MitoCheck Project Group, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. erfle@embl.de

Nature Protocols
|April 5, 2007
PubMed
Summary

This study presents a new protocol for reverse transfection of small interfering RNA (siRNA) arrays, enabling efficient genome-wide RNA interference (RNAi) screens in human cells. The method is robust, scalable, and maintains transfection efficiency for over a year.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genomics

Background:

  • Genome-wide RNA interference (RNAi) screens are powerful tools for functional genomics.
  • Existing protocols for RNAi screening can be time-consuming and require optimization for different cell types.

Purpose of the Study:

  • To develop and validate a robust, high-throughput protocol for reverse transfection of small interfering RNA (siRNA) arrays for genome-wide RNAi screens.
  • To assess the stability and efficiency of pre-fabricated siRNA arrays.
  • To demonstrate the applicability of the protocol in intact human cells using various cell lines.

Main Methods:

  • Development of a protocol for reverse transfection of cells onto 384-well siRNA arrays.
  • Automation of siRNA array production (48 arrays, 7 hours).
  • Validation using multi-channel immunofluorescence and time-lapse microscopy.
  • Testing with multiple human cell lines.

Main Results:

  • A robust protocol for reverse transfection of siRNA arrays was established.
  • Automated production of 48 arrays (384 samples each) completed in 7 hours.
  • Pre-fabricated arrays maintained transfection efficiency for at least 15 months.
  • Successful transfection demonstrated in multiple human cell lines.
  • Protocol applied to two genome-wide siRNA screens (mitosis and protein secretion).

Conclusions:

  • The developed protocol enables efficient and scalable genome-wide RNAi screening in intact human cells.
  • The protocol is cost-effective due to automated production and long-term stability of siRNA arrays.
  • This method facilitates high-throughput functional genomics studies, including the investigation of cellular processes like mitosis and protein secretion.