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

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Cell transfection is crucial for biological studies but often low-throughput, time-consuming, and error-prone, especially with multiple plasmids.
• Existing methods lack efficiency and speed, hindering large-scale genetic analysis and screening.

Purpose of the Study:

• To develop an easy, fast, and accurate high-throughput cell transfection protocol using acoustic droplet ejection (ADE) technology.
• To enable precise nanovolume delivery and multiplexing of DNA and transfection reagents in a 384-well plate format.

Main Methods:

• Utilized an acoustic droplet ejection (ADE) nanodispenser for precise nanoliter-scale delivery of DNA and transfection reagents.
• Developed a user-friendly spreadsheet-based macro to manage up to four plasmids per well from a library of 1,536 plasmids.
• Implemented a four-step protocol: diluent dispense, plasmid distribution/multiplexing, reagent dispense, and cell plating.

Main Results:

• Achieved up to 90% transfection efficiency.
• Reached nearly 100% cotransfection efficiency in multiplexing experiments.
• Demonstrated a user-friendly software-controlled process for ADE-based plasmid transfection.

Conclusions:

• The ADE-based method offers a fast, safe, and reliable approach for high-throughput cell transfection, suitable for non-specialists.
• This protocol facilitates applications like ORFeome protein expression and CRISPR-Cas9 gene function validation in non-pooled screening strategies.
• The method is adaptable to higher-scale and manual approaches, accelerating biological research.