High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Béatrice Colin; Nicolas Rocq; Benoit Deprez; Cyril Couturier

doi:10.3791/59570

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Method Article

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

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DOI:

10.3791/59570

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August 8th, 2019

Béatrice Colin¹ , Nicolas Rocq² , Benoit Deprez¹ , Cyril Couturier¹

¹Institut Pasteur de Lille, Institut National de la Santé et de la Recherche Médicale (Inserm) U1177, Drugs and Molecules for Living Systems, Université de Lille, ²L'atelier du Développeur, Marquette lez Lille

Summary

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Abstract

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

Introduction

The method presented here describes in detail how to perform DNA plasmid multiplexing and transfection in mammalian cells at high throughput using an acoustic-based liquid nanodispenser in a 384-well plate, even for nonspecialists in the field. This recently published method¹ allows performing as much as 384 independent plasmid DNA multiplexing and transfection conditions in one experiment, in less than 1 h. Single or cotransfection experiments were successful, reaching a near 100% cotransfection within the transfected cells population. This protocol makes transfection easier because most of the tedious, time-consuming, and error-prone steps ar....