Small Interfering RNA (siRNA) Transfection in Epiblast Stem Cells

Lipid-based transfection of siRNA is a technique routinely used to investigate gene function in experiments using mammalian cells cultured in vitro. Due to innate differences in cellular characteristics, the efficiency of lipid-based transfection is variable across cell types. Pluripotent cells which exist in a "primed" state such as human embryonic stem cells (hESCs) and mouse epiblast stem cells (mEpiSCs) are notorious for being refractory to lipid-based transfection systems. Herein we describe a forward transfection protocol which we routinely use to achieve upwards of 70% transfection efficiency rates in mEpiSCs. Our protocol also includes a suggested transfection timeline and details pertaining to the techniques we use to validate transfection success.