Unit

UNIT 14.11 High-Throughput Gene Silencing and mRNA Expression Analysis in Hepatocyte Sandwich Cultures

  1. Brett D. Hollingshead,
  2. Lauren M. Gauthier,
  3. Andrew D. Burdick

Published Online: 1 FEB 2013

DOI: 10.1002/0471140856.tx1411s55

Current Protocols in Toxicology

Current Protocols in Toxicology

How to Cite

Hollingshead, B. D., Gauthier, L. M. and Burdick, A. D. 2013. High-Throughput Gene Silencing and mRNA Expression Analysis in Hepatocyte Sandwich Cultures. Current Protocols in Toxicology. 55:14.11:14.11.1–14.11.11.

Author Information

  1. Drug Safety Research and Development, World Wide Research and Development, Pfizer Inc., Cambridge, Massachusetts

Publication History

  1. Published Online: 1 FEB 2013

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. Curr. Protoc. Toxicol. 55:14.11.1-14.11.11. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • hepatocyte;
  • siRNA;
  • gene expression;
  • gene silencing