Application of magnetic beads to purify cells transiently transfected with plasmids encoding short hairpin RNAs.
Short interfering (si) RNAs are commonly used to knock down expression of proteins in mammalian cells and thereby investigate protein function. siRNAs were originally introduced into mammalian cells by transient transfection of short, synthetic, double-stranded RNA oligonucleotides. More recently, a convenient, more cost-effective approach has been developed that makes use of plasmids encoding short hairpin (sh) RNAs, which are transiently or stably transfected into cells. After expression in cells, shRNAs are processed by the cell to the corresponding siRNAs. However, most protocols for transient transfection of plasmid DNAs introduce the DNA into a minority of the total cells. Therefore, to investigate the biochemical effects of protein knockdown, it is necessary to purify the transfected cells. This can be done by cotransfection of a plasmid encoding the cell surface marker protein, CD19 or CD20, followed by immunopurification of the CD19- or CD20-expressing cells with magnetic beads. The purified cells can then be used for a wide range of biochemical analyses. In addition, since the CD19/CD20 cell surface marker approach can be readily combined with analysis of cell cycle distribution of propidium iodide-stained cells, it is straightforward to determine simultaneously the biochemical and cell cycle effects of a knocked-down protein.
Adams
Peter D
PD
Fox Chase Cancer Center, Philadelphia, PA, USA.
D018941:Antigens, CD19 / Q000235:genetics; D018951:Antigens, CD20 / Q000235:genetics; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D006801:Humans; D018189:Immunomagnetic Separation; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D018941:Antigens, CD19; D018951:Antigens, CD20; D034741:RNA, Small Interfering; D012313:RNA