Establishing efficient siRNA knockdown in stem cells using fluorescent oligonucleotides.
The following protocols provide a rapid approach for establishing good working conditions for transfecting siRNAs for specific gene knockdown. By first using microscopy to evaluate efficient transfection of an inexpensive, fluorescent oligonucleotide, the researcher can later proceed with more expensive Western blot or quantitative real-time PCR (qRT-PCR) methods. Thus, the main culprit of ineffective knockdown, poor transfection, can be eliminated before engaging in tedious and time-consuming approaches for troubleshooting siRNA knockdown experiments.
Chen
Stephen W
SW
Stem Cell Group, Bioprocessing Technology Institute, Centros, Singapore.
Oh
Steve K W
SK
D000818:Animals; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism*; D055785:Gene Knockdown Techniques / Q000379:methods*; D051379:Mice; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000235:genetics*; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009841:Oligonucleotides; D034741:RNA, Small Interfering