Methods for siRNA-mediated reduction of mRNA and protein expression in human placental explants, isolated primary cells and cell lines.
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.
Forbes
K
K
Maternal & Fetal Health Research Group, School of Clinical & Laboratory Sciences, University of Manchester, St. Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK.
Desforges
M
M
Garside
R
R
Aplin
J D
JD
Westwood
M
M
D000328:Adult; D000469:Alkaline Phosphatase; D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005260:Female; D058851:GPI-Linked Proteins; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009924:Organ Culture Techniques; D011247:Pregnancy; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism*; D055815:Young Adult
D058851:GPI-Linked Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental