How it Works: Transfecting B-cell Lymphomas With siRNA

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Problem: Using small interfering RNAs (siRNAs) to exploit the mechanism of RNA interference (RNAi) has become a powerful tool to study gene function in cell culture and in vivo. These small RNA molecules are designed to target the mRNA with a complementary sequence in order to achieve degradation. Chemically synthesized 21-nucleotide siRNAs have been shown to induce RNA interference in mammalian cells. This can be useful in suppressing cancer cells like B-cell lymphomas, which are cancers derived from lymphocytes and constitute 85% of all non-Hodgkin’s lymphomas. For reasons that remain unknown, transfecting B-cell lymphomas has not been achieved with traditional techniques using lipids, chemicals and viruses. Progress has been made with electroporation, but this process results in a high rate of cell death.

Solution: Jessica Alluin, a Research Associate in the Department of Molecular Biology at The City of Hope, used Bio-Rad’s Gene Pulser MXcell electroporation system to successfully transfect two cell lines of B-cell lymphomas with siRNA against the HPRT1 gene. The Gene Pulser MXcell electroporation system is designed to ensure highly efficient delivery of molecules such as siRNA into a variety of mammalian cells, including primary and difficult-to-transfect cells.

Critical to the success of any electroporation experiment is the ability to optimize experimental conditions. This leads to increased transfection efficiency while maintaining cell viability and the need for less precious sample. Unlike other electroporation systems, every critical parameter is programmable with Bio-Rad’s Gene Pulser, from waveform to number of pulses, enabling researchers to easily find the ideal set of conditions for their specific cells. Alluin appreciated the Gene Pulser’s distinctive ability to vary and test multiple electroporation conditions on one plate, fine tuning for both transfection efficiency and cell viability.

Specifically, she suspended B-cell lymphoma cell lines JEKO-1 and SUDHL-6 cells in Gene Pulser electroporation buffer and divided into sets of two: one control and one experimental. Each set consisted of buffer and cells whereas the experimental tube had the addition of HPRT1 synthetic siRNA to determine RNAi knockdown. Each set was separated into the wells of a 24-well electroporation plate and electroporated with the Gene Pulser MXcell system using a resistance of 1,000Ω, varying the waveform, voltage, capacitance or duration and number of pulses to determine the optimal condition for each cell type. The electroporated cells were transferred into a 6-well plate and incubated at 37°C.

At 48 hours post-transfection, aliquots were taken from each set of transfected cells to determine cell viability under a microscope. Cells with a viability of 50% or greater were processed to determine HPRT1 mRNA expression levels and therefore the transfection efficiency. The Gene Pulser MXcell electroporation system proved to be effective in transfecting B-cell lymphomas once the conditions resulting in minimal cell death were identified. Electroporation conditions 16 and 9 demonstrated the best siRNA delivery methods in JEKO-1 and SUDHL-6, respectively, with minimal cell death.

A comprehensive set of electroporation protocols for mammalian cell lines submitted by scientists worldwide is available through Bio-Rad’s website at www.biorad.com/transfectionprotocols

Categories: How it Works

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The Online Lab Manager

Published: January 1, 2010

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