Establishing efficient siRNA knockdown in mouse embryonic stem cells.
FAM-labeled oligo dT (FAMdT) was utilized as a means to gauge efficient transfection of small nucleic acids into mouse embryonic stem cell (mESC) colonies. Using colonies grown overnight, transfection was restricted largely to the periphery of the colonies with only a 40% decrease in Oct-3/4 RNA transcript levels following cognate Oct-3/4, small interfering RNA (siRNA) delivery. However, transfection of mESC 4 h after seeding gave greater than 90% cells being successfully transfected based on quantitative real-time PCR detection of approximately 90% Oct-3/4 RNA transcript knockdown. This method provides an economical and efficient means by which to determine effective transfection conditions, and establish efficient siRNA knockdown of reportedly difficult to transfect cell lines such as mESC.
Chen
Stephen
S
Stem Cell Group, Bioprocessing Technology Institute, 20 Biopolis Way, Singapore, Singapore.
Choo
Andre
A
Wang
Nai-Dy
ND
Too
Heng-Phon
HP
Oh
Steve Kah Weng
SK
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*
D050814:Octamer Transcription Factor-3; D034741:RNA, Small Interfering