Bio-Rad Gene Pulser MXcell™ Electroporation System - Manual (Page 52)

Factors Affecting Electroporation
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Although transformation of most cell types has been accomplished using plasmid DNA
isolated by a variety of methods, the sample purity has an effect on transformation
efficiency. Significantly lower transformation efficiencies are generated with unpurified
plasmid DNA than with purified plasmid DNA. Plasmid produced using the Bio-Rad Aurum
matrix is as efficient as CsCl-purified plasmid for transformation of mammalian cells.
However, as long as plasmids used for electroporation are all prepared in the same manner,
changes in expression levels are due to differences in transcription or translation of the
gene of interest. The concentration range for plasmid DNA electroporation is typically 5 to
40 μg/ml, and is dependent of the cell type used.
SI RNA
The quality of siRNAs can significantly influence the outcome of siRNA transfections and
RNAi experiments. The siRNAs should be free of reagents carried over from synthesis. Also,
dsRNA contaminants larger than approximately 30 bp cause cytotoxicity. In addition,
undesired off-target effects can be avoided by using highly purified siRNAs.
The optimal siRNA concentration and its capacity for gene silencing are influenced in part
by properties of the target gene including the following: mRNA localization, stability, and
abundance; and target protein stability and abundance. If too much siRNA is used in
electroporation, it may be toxic. Conversely, if too little siRNA is transfected, reduction of
target gene expression may be undetectable. The optimal amount must be determined
empirically by varying the siRNA amount within a limited range. We recommend using
siRNAs at concentrations of 10 to 100 nM.
Experiments involving siRNAs have been mostly limited to immortalized cell lines, because
these cells are relatively easy to grow, maintain, and transfect. However, primary cells are
often a preferable model for studying gene function because they are more similar to their in
vivo counterparts than immortalized cell lines. Electroporation provides a highly efficient
method for direct transfer of siRNAs into primary cells.
Impurities in siRNA oligonucleotide preparations can reduce the potency and delivery
efficiency of siRNAs, and can increase the risk of toxicity in gene silencing experiments.
Using high quality duplex siRNA, siLentMer™ Dicer-Substrate siRNA duplexes will lead to
improved success rates and reproducibility of gene silencing experiments.
Electroporation Buffer
The electroporation medium influences cell viability and transfection efficiency in several
ways. The osmotic balance of the electroporation buffer, the choice of salt, and the
requirements of specific ions all play a role in electroporation. In general, a media that will
mimic the natural cytoplasmic composition of the cell, such as Gene Pulser electroporation
buffer is recommended.
The buffer used to electroporate mammalian cells has a direct effect on the time constant,
since the sample resistance (R) is mainly due to the buffer ionic strength. The buffer
components also influence transfection efficiency and cell viability. Traditionally, a buffer
with high ionic strength (low resistance) such as PBS is used when electroporating
mammalian cells at a high capacitance. Serum-free growth medium has also been routinely
used in electroporation. The volume of liquid/buffer in the electroporation well has a
significant effect on sample resistance, and is inversely proportional to the volume of the
buffer used.