High‐Throughput Gene Silencing and mRNA Expression Analysis in Hepatocyte Sandwich Cultures

Brett D. Hollingshead1, Lauren M. Gauthier1, Andrew D. Burdick1

1 Drug Safety Research and Development, World Wide Research and Development, Pfizer Inc., Cambridge, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.11
DOI:  10.1002/0471140856.tx1411s55
Online Posting Date:  February, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Reverse Transfection of Primary Mouse Hepatocytes
  • Basic Protocol 2: RNA Isolation, cDNA Preparation, and Gene Expression
  • Support Protocol 1: Considerations for Gene Turnover Rate and Treatment Methods of Transfected Hepatocytes
  • Support Protocol 2: Real‐Time PCR Data Analysis Considerations
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Reverse Transfection of Primary Mouse Hepatocytes

  Materials
  • Dharmacon siRNA on‐target and control complexes (or equivalent)
  • siRNA complexing medium (see recipe)
  • Hepatocyte dilution medium (see recipe)
  • Hepatocyte growth medium (see recipe)
  • DharmaFECT 4 transfection reagent (Thermo Scientific)
  • Hepatocytes
  • Collagen‐coated 96‐well cell culture dishes
  • Matrigel (BD Biosciences)
  • UV spectrometer
  • 37°C water bath
  • 1.5‐, 15‐, and 50‐ml sterile polypropylene tubes
  • Hemacytometer or automated cell counter
  • Sterile, disposable 50‐ml reagent troughs
  • 8‐well, 200‐µl capacity multichannel pipettor (12‐well multichannel pipettor, optional)
  • 37°C humidified cell culture incubator
  • 75‐ml sterile, polypropylene plastic Erlenmeyer flask
  • 8‐ or 12‐well aspirating attachment for a cell culture waste vacuum (optional)

Basic Protocol 2: RNA Isolation, cDNA Preparation, and Gene Expression

  Materials
  • Cultured hepatocytes in culture plates
  • Qiagen RNeasy 96 Universal BioRobot 8000 kit (Qiagen, cat. no. 967152), for automated extraction or Qiagen RNeasy 96 kit (Qiagen, cat. no. 74181) or Promega SV 96 total RNA isolation system (Promega, cat. no. Z3505), for manual RNA extraction
  • RNA to cDNA reverse transcription reagents (e.g., Applied Biosystems High‐Capacity cDNA reverse transcription kit)
  • Nuclease‐free water
  • Real‐time PCR master mix (e.g., TaqMan Universal PCR master mix, Invitrogen)
  • Real‐time PCR gene‐specific amplification primers and probes (e.g., TaqMan Gene Expression Assays, Invitrogen)
  • 8‐well, 10‐ to 20‐µl and 200‐µl capacity multichannel pipettor
  • Sterile, nuclease‐free barrier pipet tips (10‐, 200‐, and 1000‐µl)
  • 50‐ml sterile, nuclease‐free disposable reagent troughs
  • Qiagen Universal Biorobot System (automated) or a 96‐well compatible vacuum manifold (manual) (e.g., Promega Vac‐Man 96 vacuum manifold)
  • NanoDrop spectrophotometer (or similar), optional
  • 96‐well optical thermal cycler plates (specific for instrument being used)
  • 96‐well plate sealing optical film and sealer
  • Swinging bucket centrifuge with a 96‐well plate rotor
  • Standard 96‐well compatible thermal cycler
  • Real‐time PCR thermal cycler (e.g., ABI 7900 Real Time PCR System)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Anderson, E.M., Birmingham, A., Baskerville, S., Reynolds, A., Maksimova, E., Leake, D., Fedorov, Y., Karpilow, J., and Khvorova, A. 2008. Experimental validation of the importance of seed complement frequency to siRNA specificity. RNA 14:853‐861.
   Davila, J.C. and Morris, D.L. 1999. Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes. In Vitro Cell Dev. Biol. Anim. 35:120‐130.
   Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811.
   Jackson, A.L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo, J., Johnson, J.M., Lim, L., Karpilow, J., Nichols, K., Marshall, W., Khvorova, A., and Linsley, P.S. 2006. Position‐specific chemical modification of siRNAs reduces “off‐target” transcript silencing. RNA 12:1197‐1205.
   Wong, M.L. and Medrano, J.F. 2005. Real‐time PCR for mRNA quantitation. Biotechniques 39:75‐85.
Internet Resources
  http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasy96universaltissue8000.aspx#Tabs=t2
  This link guides the user to the automated RNA extraction process mentioned in Basic Protocol 1.
  http://www.promega.com/resources/protocols/technical‐bulletins/0/sv‐96‐total‐rna‐isolation‐system‐protocol/
  This link guides the user to one of the manual RNA extraction processes mentioned in Basic Protocol 2.
  http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_053906.pdf
  This is an application note from Applied Biosystems explaining the basics of real‐time PCR and determination if Ct values.
  http://www.bioconductor.org/packages/2.10/bioc/vignettes/ddCt/inst/doc/rtPCR.pdf
  This document discusses in detail the ddCt method for real‐time PCR data analysis.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library