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

K. Forbes, M. Desforges, R. Garside, J.D. Aplin, and M. Westwood
Source: Placenta
Publication Date: (2009)
Issue: 30(2): 124-129
Research Area:
Stem Cells
Cells used in publication:
Species: human
Tissue Origin:
Nucleofector® I/II/2b
BeWo: Cells destined for transfection using an electroporation-based system - nucleofection, were grown in T75 flasks until 70–80% confluent, then trypsinised and counted. 1 Ü? 106 cells/condition were resuspended in cell line solution L or cell line solution V and transfected following the manufacturers' instructions using programs A-020, T-020, X-001 and X-005 (Amaxa Biosystems, Germany). Following transfection, cells were plated at approximately 7 Ü? 10^4/well for 24-well plates and approximately 3 Ü? 10^5/well for 6 well plates. Primary cytotrophoblast cells: Cells intended for transfection by nucleofection were centrifuged at 1000 g for 5 min immediately after isolation and then resuspended in the nucleofection buffer (100 μl/2 Ü? 106 cells) provided in the basic primary mammalian epithelial cell nucleofector kit (Amaxa Biosystems, Germany). 2 Ü? 10^6 cells/condition were transfected using programme Nucleofector programmes S-005, T-020, T-023 or U-017 and then plated onto 12 well plates at 1–2 Ü? 10^6 cells/well in 1.5 ml culture medium (DMEM/Ham's F12 (1:1, v/v) containing 10% fetal calf serum and antibiotics). 1st trimester human placenta explants: In order to carry out transfection by nucleofection, 2–3 pieces of placental tissue were placed in 100 μl basic primary epithelial cell nucleofector buffer containing siRNA or vehicle. Using a wide-ended 1.5 ml Pasteur pipette, tissue and buffer were transferred to a Nucleofector cuvette and then exposed to programs X005, X001, U006, U007 and U017 before removal using pipettes supplied with the Nucleofector kit.
The use of RNA interference (RNAi) to deplete individual proteins from cells or tissue has revolutionised our ability to characterise gene function. The placenta is an attractive target for studies in which the role of specific proteins can be compared with cell culture models and explanted villous tissue where physiological function can be maintained ex vivo. In this study, we compared a variety of commercially available reagents and approaches to define methods for efficient delivery of siRNA to placental cells. Protocols optimised using fluorescently-labelled siRNA were subsequently tested using siRNA sequences that target placental alkaline phosphatase (PLAP), chosen because of its high abundance in trophoblast. mRNA abundance was assayed using qRT-PCR, and the effect on protein was examined using immunolocalisation. We report that different protocols are required for BeWo choriocarcinoma cells (nucleofection), primary cytotrophoblast cells (lipid-based transfection) and villous tissue explants (nucleofection). The results provide guidelines for optimal siRNA-mediated knockdown in these three models of the human placenta.