title: High-throughput gene silencing and mRNA expression analysis in hepatocyte sandwich cultures.
abstract: Primary hepatocyte sandwich cultures are useful for a variety of research applications where maintenance of metabolic competency is essential. To ensure an optimal hepatocellular phenotype, cells are seeded on collagen-coated dishes and embedded with an overlay of Matrigel. This culturing condition makes gene silencing by traditional reagent-mediated transfection methods challenging. Here, an siRNA delivery method in primary mouse hepatocytes that allows cells to be cultured with Matrigel overlay is described. This method delivers >80% mRNA silencing with minimal alterations in cell viability. A 96-well format allows for high-throughput RNA processing and downstream quantitative PCR applications and reduces time and resources. This format is particularly useful when experiments requiring many different sampling conditions (such as pharmacologic dose-response curves) are required.
authors: Drug Safety Research and Development, World Wide Research and Development, Pfizer Inc., Cambridge, Massachusetts, USA.
authors: Brett D
authors: BD
authors: Hollingshead
authors: Lauren M
authors: LM
authors: Gauthier
authors: Andrew D
authors: AD
authors: Burdick
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020868:Gene Silencing; D022781:Hepatocytes / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D051379:Mice; D061251:Primary Cell Culture / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering