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High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology
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Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates.

Holger Erfle1, Beate Neumann, Phill Rogers

  • 1Cell Biology/Biophysics Unit, EMBL, Heidelberg, Germany. holger.erfle@bioquant.uni-heidelberg.de

Journal of Biomolecular Screening
|July 5, 2008
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Summary

A new protocol enables automated solid-phase reverse transfection in multiwell plates, allowing for RNAi screening and gene knockdown analysis. This method offers cost-efficiency and flexibility for multiplexing cellular assays.

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Area of Science:

  • Cell biology
  • Molecular biology
  • Biotechnology

Background:

  • Solid-phase reverse transfection on cell microarrays is a high-throughput method for parallel mammalian cell transfection.
  • Current methods are limited to microscopy-based analyses, restricting downstream applications like RT-PCR.
  • Higher cell numbers for statistical robustness in microscopy are not feasible with current array-based methods.

Purpose of the Study:

  • To develop a quick and reliable protocol for automated solid-phase reverse transfection of human cells with siRNAs in multiwell plates.
  • To complement existing solid-phase reverse transfection on cell microarrays with a multiwell plate format.
  • To enable downstream analyses such as RT-PCR and improve statistical power through increased cell numbers.

Main Methods:

  • Developed an automated solid-phase reverse transfection protocol using multiwell plates.
  • Tested the protocol for RNAi-mediated gene knockdown in U20S, RPE1, A549, and HeLa cell lines.
  • Assessed transfection efficiency using RT-PCR and nuclear phenotyping via fluorescence microscopy.

Main Results:

  • The new protocol is quick, reliable, and automatable.
  • Solid-phase reverse transfection in multiwell plates retains advantages like long-term storage, reduced cytotoxicity, and lower cost per screen.
  • Transfection efficiency remained high even 3 months after plate fabrication, as confirmed by RT-PCR and microscopy.

Conclusions:

  • Solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays.
  • This method overcomes limitations of array-based transfection, enabling broader downstream analyses.
  • The protocol supports high-throughput screening and gene knockdown studies with enhanced statistical power.