Analysis of Gene Expression and Gene Silencing in Human Macrophages

Fernando O. Martinez1

1 Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.28
DOI:  10.1002/0471142735.im1428s96
Online Posting Date:  February, 2012
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Abstract

This unit describes how to execute a gene expression study with human macrophages. It includes protocols for human macrophage preparation, RNA extraction, real‐time PCR analysis, and microarray analysis. The unit also includes a protocol for gene silencing in human macrophages. Altering gene expression can be useful to study the contribution of the gene to macrophage function or even expression of other genes. Curr. Protoc. Immunol. 96:14.28.1‐14.28.23. © 2012 by John Wiley & Sons, Inc.

Keywords: buffy coat; monocyte; macrophage; microarray; transcriptome; real‐time PCR; SYBR Green RT PCR; siRNA

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Monocyte Isolation from Human Peripheral Blood
  • Basic Protocol 2: Stimulation of Monocytes to Induce Macrophage Maturation
  • Support Protocol 1: RNA Isolation
  • Basic Protocol 3: Using Real‐Time PCR to Study Gene Expression
  • Basic Protocol 4: Using Microarrays to Study Whole‐Genome Gene Expression
  • Basic Protocol 5: Macrophage Gene Expression Knock‐Down with siRNA Duplexes
  • Reagents and Solution
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Monocyte Isolation from Human Peripheral Blood

  Materials
  • Peripheral blood ( appendix 3F)
  • Phosphate‐buffered saline (PBS; no calcium or magnesium; appendix 2A)
  • Ficoll‐Paque PLUS (GE Healthcare, cat. no. 17–1440‐02); other companies produce Ficoll, but this version is endotoxin free, which is vital to avoid monocyte activation
  • Complete RPMI medium (see recipe)
  • 46% Percoll (285 mOsm; see recipe)
  • 50‐ml polypropylene tubes (e.g., BD Falcon)
  • Centrifuge
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion ( appendix 3B)
NOTE: Monocytes adhere to polystyrene tubes, which decreases the yield of the protocol. It is recommended to use polypropylene tubes throughout the procedure. The procedure is preferably carried out at room temperature, but it can be done at 4°C.NOTE: This procedure is designed for 50 ml of fresh blood or one buffy coat bag. If smaller volumes are used, scale reagents appropriately. Unless specified, the procedure is the same for fresh blood or buffy coat bags.

Basic Protocol 2: Stimulation of Monocytes to Induce Macrophage Maturation

  Materials
  • Peripheral blood donor (see appendix 3F; optional)
  • X‐VIVO 10 medium (BioWhittaker)
  • Complete RPMI medium (see recipe)
  • Complete OptiMEM medium (see recipe)
  • Peripheral blood monocytes (see protocol 1)
  • Recombinant human M‐CSF, (PeproTech, cat. no. 300–25)
  • Recombinant human GM‐CSF, (PeproTech, cat. no. 300–03)
  • Vacutainer SST tubes (Becton Dickinson; optional)
  • Centrifuge
  • Water bath at 56°C (optional)

Support Protocol 1: RNA Isolation

  Materials
  • Macrophages growing in culture (see protocol 2)
  • Dulbeccoapos;s phosphate‐buffered saline (DPBS; with calcium and magnesium; e.g., Invitrogen)
  • RNeasy Mini Kit (Qiagen, cat. no. 74104) including:
    • Buffer RLT
    • RNeasy columns
    • 2‐ml collection tubes
    • Buffer RW1
    • Buffer RPE
    • Buffer RDD
  • 70% ethanol
  • Molecular‐biology‐grade H 2O
  • RNase‐free DNase set (Qiagen, cat. no. 79254; optional; to be used for real‐time PCR samples if required)
  • 2.5% agarose gel in 1× TBE buffer (prepared as described in unit 10.4)
  • 0.1% (w/v) ethidium bromide
  • Formamide, deionized (see recipe)
  • Cell scrapers
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 65°C heat block
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4)

Basic Protocol 3: Using Real‐Time PCR to Study Gene Expression

  Materials
  • Reverse transcription kit: SuperScript III First‐Strand Synthesis System for RT‐PCR (Invitrogen, cat. no. 18080–051) including:
    • 50 µM oligo(dT)
    • 10 mM dNTP mix
    • RNase‐free H 2O
    • 10× RT buffer
    • M DTT
    • 40 U/µl RNaseOUT
    • 200 U/µl SuperScript III reverse transcriptase
    • 2 U/µl E. coli RNase H
  • RNA from cultured monocytes (mature macrophages) (see protocol 3)
  • SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4309155)
  • Thermal cycler
  • Thin‐walled PCR tubes
  • Machine‐specific optical tubes and caps for PCR machine

Basic Protocol 4: Using Microarrays to Study Whole‐Genome Gene Expression

  Materials
  • A computer with Internet access is required
  • Two pieces of software must be downloaded:
    • R‐Bioconductor (Ihaka and Gentleman, ; see Internet Resources)
    • TMeV MultiExperiment Viewer (Saeed et al., ; see Internet Resources)
  • The following Web tools will also be used:
    • DAVID‐Bioinformatics Resources (Huang da et al., ; see Internet Resources)
    • NCI‐Nature Pathway Interaction Database (Schaefer et al., ; see Internet Resources)
Microarray Analysis FlowchartRegardless of the microarray choice, the service will provide a table with gene‐expression information. Generally, the rows represent the genes and every column corresponds to a sample. This is an expression matrix of X experiments versus Y genes, where X is the number of samples and Y is the number of genes in the array. The service should provide normalized values and information about whether the values are raw or Log2 transformed. Other information, describing the meaning of every column and quality of the microarray experiment, should be included in their report. All service providers can help with the analysis; however, having the skills to process the samples will provide more information and understanding. Below, we describe five operations required to convert the expression matrix into meaningful information (see Fig. ).

Basic Protocol 5: Macrophage Gene Expression Knock‐Down with siRNA Duplexes

  Materials
  • Gene‐specific Stealth RNAi siRNA (Invitrogen): for design, see Strategic Planning
  • Stealth RNAi siRNA negative control kit (Invitrogen, cat. no. 12935–100; the kit contains low, medium, and high‐GC Scrambled Stealth RNAi siRNA negative control duplexes)
  • OptiMEM medium, serum‐free (Invitrogen, cat. no. 11058–021
  • INTERFERin (Polyplus, cat. no. 409–10; http://www.polyplus‐transfection.com/; 1 ml of INTERFERin is sufficient to perform 500 to 1000 transfections in 24‐well plates)
  • 24‐well tissue culture plates
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Figures

  •   FigureFigure 14.28.1 Schematic representation of the Ficoll/Percoll monocyte isolation protocol.
  •   FigureFigure 14.28.2 Representative amplification and disassociation curves in a real‐time PCR reaction. Panel A shows prototypic amplification plots of four different samples—the lines represent samples with high gene expression (green), medium expression (blue), low expression (orange), and absent expression (pink). Panel B shows a dissociation curve; the analysis is performed on the real‐time PCR products and helps to evaluate the specificity of your primers and corroborate the presence of one amplicon, represented by a unimodal curve. Data are obtained by slowly increasing the temperature from 60° to 95°C while collecting fluorescence data. The increase in temperature causes PCR products to denature, a process accompanied by a decrease in fluorescence for solutions containing SYBR Green I dye.
  •   FigureFigure 14.28.3 Flowchart for microarray analysis. The figure shows a flowchart for microarray analysis, with the main techniques used indicated. In the green boxes to the left, the level at which differences are evaluated is indicated. On the right are shown icons of representative results. At image level, pre‐processing and sample normalization are required. The inset shows representative box plots and histograms, before and after normalization. At the sample level, hierarchical clustering (HCL) and principal component analysis (PCA) help to estimate global differences. The inset shows a prototypic HCL and PCA graph. At gene level, statistical analysis followed by clustering analysis permits identification and organization of the changed genes. The inset shows a four clusters K means clustering and an HCL applied to the gene level. After selecting and grouping genes, the pathway overrepresentation can be studied. The inset shows a fragment of a DAVID.ease output table.
  •   FigureFigure 14.28.4 Monocyte isolation control by FACS or microscopy. Panel A shows a representative FACS plot after Percoll isolation. Minimal contamination by lymphocytes should be observed. Panel B shows a cytospin of a typical preparation stained with methylene blue plus eosin. By microscopy lymphocytes appear with a round, darker, and small nucleus; monocytes instead are characterized by a kidney‐shaped, irregular, and lighter nucleus.
  •   FigureFigure 14.28.5 Appearance of intact total RNA by gel electrophoresis and Agilent Bioanalyzer. Panel A shows a gel electrophoresis of intact total RNA. Arrows indicate the position of 18S and 28S ribosomal RNA bands. Panel B shows an Agilent Bioanalyzer electrophoretic profile where the ribosomal peaks 28S/18S should show a ratio close to 2.0, and the baseline is very low. The RNA integrity number (RIN) is a value provided by the Agilent Bioanalyzer software to estimate the integrity of total RNA samples. A RIN number of 10 indicates an intact RNA; a RIN of 8 is acceptable for gene expression analysis.

Videos

Literature Cited

   Blaschke, E., Eklund, A., Skog, S., and Danielsson, B. 1985. Isolation of human alveolar macrophages and lymphocytes from bronchoalveolar lavage fluid by centrifugal elutriation. Scand. J. Clin. Lab. Invest. 45:691‐696.
   Colotta, F., Borré, A., Wang, J.M., Tattanelli, M., Maddalena, F., Polentarutti, N., Peri, G., and Mantovani, A. 1992. Expression of a monocyte chemotactic cytokine by human mononuclear phagocytes. J. Immunol. 148:760‐765.
   Du, P., Kibbe, W.A., and Lin, S.M. 2008. Lumi: A pipeline for processing Illumina microarray. Bioinformatics 24:1547‐1548.
   Fleetwood, A.J., Dinh, H., Cook, A.D., Hertzog, P.J., and Hamilton, J.A. 2009. GM‐CSF‐ and M‐CSF‐dependent macrophage phenotypes display differential dependence on type I interferon signaling. J. Leukoc. Biol. 86:411‐421.
   Gautier, L., Cope, L., Bolstad, B.M., and Irizarry, R.A. 2004. Affy—Analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20:307‐315.
   Helming, L., Tomasello, E., Kyriakides, T.R., Martinez, F.O., Takai, T., Gordon, S., and Vivier, E. 2008. Essential role of DAP12 signaling in macrophage programming into a fusion‐competent state. Sci. Signal. 1:ra11.
   Huang da, W., Sherman, B.T., and Lempicki, R.A. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4:44‐57.
   Ihaka, R., and Gentleman, R. 1996. A language for data analysis and graphics. J. Comp. Graph. Stat. 5:299‐314.
   Irizarry, R.A., Bolstad, B.M., Collin, F., Cope, L.M., Hobbs, B., and Speed, T.P. 2003. Summaries of affymetrix GeneChip probe level data. Nucleic Acids Res. 31:e15.
   Krausgruber, T., Blazek, K., Smallie, T., Alzabin, S., Lockstone, H., Sahgal, N., Hussell, T., Feldmann, M., and Udalova, I.A. 2011. IRF5 promotes inflammatory macrophage polarization and TH1‐TH17 responses. Nat. Immunol. 12:231‐238.
   Martinez, F.O., Gordon, S., Locati, M., and Mantovani, A. 2006. Transcriptional profiling of the human monocyte‐to‐macrophage differentiation and polarization: New molecules and patterns of gene expression. J. Immunol. 177:7303‐7311.
   Martinez, F.O., Helming, L., and Gordon, S. 2009. Alternative activation of macrophages: An immunologic functional perspective. Annu. Rev. Immunol. 27:451‐483.
   Saeed, A.I., Bhagabati, N.K., Braisted, J.C., Liang, W., Sharov, V., Howe, E.A., Li, J., Thiagarajan, M., White, J.A., and Quackenbush, J. 2006. TM4 microarray software suite. Methods Enzymol. 411:134‐193.
   Schaefer, C.F., Anthony, K., Krupa, S., Buchoff, J., Day, M., Hannay, T., and Buetow, K.H. 2009. PID: The pathway interaction database. Nucleic Acids Res. 37:D674‐D679.
Internet Resources
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