Abstract
Cell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in “hard-to-transfect” MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs.
Highlights
Multiple myeloma (MM) is a cancer affecting terminally differentiated plasma B cells [1]
Transfection efficiency is judged by introduction of an expression plasmid for EGFP (Fig. 1a), and – dependent on the human MM cell lines (HMCLs) – transfection efficiencies of up to 40% can be achieved with standard gear electroporations (Table 1)
Either cell sorting for EGFP-positive cells, or co-electroporation of an expression plasmid for CD4D [21] and subsequent microbead selection of CD4-positive cells can be employed to obtain highly enriched fractions of strongly transfected cells (Fig. 1d, see Methods section for an exact description of the purification steps). These cells can be used in experiments involving their actual payloads, for example cotransfected shRNA expression vectors [21,23,24,25], and they are suitable for transient knockdown studies in applications such as apoptosis induction, drug testing, proliferation assessments or Western blotting
Summary
Multiple myeloma (MM) is a cancer affecting terminally differentiated plasma B cells [1]. We have over the past ten years successfully used transient transfection of HMCLs with pSUPER short hairpin RNA expression vectors via electroporation [21,22,23,24,25]. The necessity for purification adds to the amount of work-time needed, potentially increases the stressfulness of the whole methodology and increases the overall cost of the procedure. This method can in principle be scaled up at will, it is in practice rather cumbersome to isolate high numbers We tested the efficiency of knockdown approaches using the same electroporation conditions but employing siRNA or stealth siRNA oligonucleotides instead of short-hairpin expression vectors
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