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Tutorials : Dec 1, 2007 ( )
Optimization of Electroporation Parameters
Using Eppendorf's Multiporator to Introduce siRNA into Jurkat Cells!--h2>
RNA interference (RNAi), an important tool for gene expression and gene function analyses, can be induced by the introduction of sequence-specific small interfering RNAs (siRNAs) into cells. For an accurate analysis of RNAi experiments, a high rate of positively transfected cells exhibiting a gene silencing effect is required. Electroporation is an efficient transfection method; several studies have shown that this technique can be successfully used to bring siRNA into cells.
It is not sufficient, however, to focus only on the transfection rate; scientists must also take the survival rate of the cells and the reproducibility of the experiment into account in order to generate reliable data.
For this reason, it is important to optimize transfection conditions carefully. The electrical parameters as well as culture conditions, design, and concentration of nucleic acid introduced all have an effect on the success of the experiment. As such, time-intensive optimization steps may be necessary until sufficient results can be achieved. Therefore, it is advantageous to choose an electroporation system that enables easy optimization and is also able to reproduce suitable parameters in a reliable way.
The Eppendorf (www.eppendorf.com) Multiporator®, together with the specially designed Hypoosmolar Electroporation Buffer, creates a system for effectively transfecting eukaryotic cells without severely damaging them. The application of extremely short pulses, in the microsecond range (soft pulses), ensures high survival rates of the cells.
Additionally, the pulses are electronically regulated, which makes the parameters independent from the resistance properties of the sample and allows uniform and reproducible pulses.
The low-conductive buffer system markedly reduces the current flow, and the electrically induced pores are much larger than those obtained from pulses in conductive solutions. The components of the media are adapted to the cytosolic ion composition of the cell, which prevents the cellular Na+/K+ gradient from collapsing during electroporation. The low osmolarity of the hypoosmolar buffer lets the cells swell up and round off, thus enabling an easier and more controlled electroporation.
Crucial parameters for successful electroporation are the voltage, the length of the pulse (time constant, t), and the number of pulses used. The voltage, which has to be set on the device, depends on the cell type (cell diameter), gap width of the cuvette, and the temperature. It is advisable to carry out a series of experiments employing different values. Ideal pulse lengths for electroporation have proven to be 40–100 µs at room temperature and 15–40 µs at 4°C.
For most cell lines, electroporation is carried out with one pulse. If one pulse proves to be insufficient, two or more pulses may be used. During multiple pulsing operations, the Multiporator automatically maintains a 60-second interval between pulses to allow the cell membrane to regenerate.
Transferring siRNA into Jurkat Cells
In order to determine the appropriate electroporation parameters for gene silencing experiments, several analyses were conducted. In the first experiment, different combinations of electrical parameters were tested with fluorescence-labeled siRNA molecules, which allow fast and easy detection using a fluorescence microscope or a FACS. Jurkat cells were resuspended in the hypoosmolar buffer and were mixed with Alexa 488-labeled control siRNA (Qiagen). The cells were electroporated in the Multiporator by a single pulse using the different parameters.
The electroporation conditions with the strongest fluorescence signal intensity and the lowest cytotoxicity were revealed by FACS analysis (Figure 1).
For the next step, a functional siRNA duplex targeting the cyclin-dependent kinase Cdc2 was used in various amounts. The gene silencing effect was measured by RT-PCR with isolated total RNA in order to determine the minimal amount of specific siRNA necessary for the experiments.
The final gene silencing experiment involved the electroporation of Jurkat cells with two specific functional Cdc2 siRNA molecules using optimized electroporation parameters and 1.0 µg of siRNA, which was shown to be most suitable in the previous approach (Figure 2).
The cells were transfected in triplicate with two Cdc2-specific siRNA and a nonsilencing control siRNA. The Cdc2 mRNA expression was analyzed by quantitative RT-PCR. As seen in Figure 3, the replicates of both Cdc2-specific siRNAs induced significant gene silencing of the target mRNA (up to 85%) in Jurkat cells.
The efficient transfer of siRNA and a high survival rate of the cells is a prerequisite for a gene silencing approach to be considered successful. Multiporator’s suitability for this work derives from its balanced system consisting of the device and an electroporation buffer. Electronic pulse regulation makes the optimization process fast and easy and enables high reproducibility since electric parameters remain stable. Multiporator, therefore, requires just a few optimization steps to establish a functional and reliable experiment with silencing efficiencies of up to 85% in Jurkat cells.
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