Methods for siRNA-mediated Reduction of mRNA and Protein Expression in Human Placental Explants, Isolated Primary Cells and Cell Lines

Delivery of siRNA to BeWo cells. 24h after transfection cells were fixed and stained with DAPI (blue) (A); optimal delivery of fluorescently-labelled siRNA (red) was achieved using an Amaxa Nucleofector with cell line solution L and program X005 (B). (C) Cells exposed to transfection conditions in the absence of siRNA (mock) or cells transfected with non-targeting (100nM) or PLAP-specific siRNA (100nM) were cultured for 72h and then analysed for PLAP mRNA expression (median and range, n=7). Raw data were analysed by using the Kruskal–Wallis test (*p<0.05 versus control cells) and are presented as median and range mRNA expression relative to the control (untransfected) sample for the corresponding experiment. Expression of PLAP protein (green) was analysed 72h after transfection with non-targeting (D) or PLAP-specific siRNA (E); nuclei are stained with PI (red).

Delivery of siRNA to primary term cytotrophoblast cells. 18h after isolation, primary cytotrophoblast cells were transfected with fluorescent-labelled siRNA (red) and then 48h later, cells were fixed and stained with DAPI (blue). Images show distribution of siRNA in control cells (A) or cells transfected with DharmaFECT2 (B). (C) Mock-transfected (DharmaFECT2) cells or cells transfected with non-targeting (100nM) or PLAP-specific siRNA (100nM) were cultured for 48h and then analysed for PLAP mRNA expression (n=5). Raw data were analysed by using the Kruskal–Wallis test (*p<0.05 versus control cells) and are presented as median and range mRNA expression relative to the control (untransfected) sample for the corresponding experiment. Expression of PLAP protein (green) was analysed 48h after transfection with non-targeting (D) or PLAP-specific siRNA (E); nuclei are stained with PI (red).

Delivery of siRNA to first trimester placental explants. 24h after transfection tissue was fixed and stained with DAPI (blue). Images show distribution of fluorescently-labelled siRNA (red) in (A) control tissue (no transfection reagents) and (B) following transfection with the Amaxa Nucleofector (basic mammalian epithelial cell solution; program U007). Arrows indicate cytotrophoblast cells (CT), syncytium (ST) and stroma. (C) Tissue exposed to transfection conditions in the absence of siRNA (mock) or tissue transfected with non-targeting (100nM) or PLAP-specific siRNA (100nM) was cultured for 72h and then analysed for PLAP mRNA expression (n=4). Raw data were analysed by using the Kruskal–Wallis test (*p<0.05 versus control cells) and are presented as median and range mRNA expression relative to the control (untransfected) sample for the corresponding experiment. Expression of PLAP protein (green) was analysed 48h after transfection with non-targeting (D) or PLAP-specific siRNA (E); nuclei are stained with PI (red).