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Example of Use of TaqMan Real‐Time RT‐PCR to Analyze Bacterial Gene Transcript Levels: Haemophilus influenzae

Jason W. Johnston1

1University of Kentucky, Lexington, Kentucky

Publication Name: 
Current Protocols in Microbiology
Unit Number: 
Unit 1D.1
DOI: 
10.1002/9780471729259.mc01d01s15
Online Posting Date: 
November, 2009
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Jason Johnston

Abstract

The protocol in this unit describes the steps for gene expression analysis of Haemophilus influenzae in broth culture. Steps for hot acid phenol RNA extraction, RNA cleanup, quantitation, assessment, and preparation for TaqMan qRT-PCR are described. The validation, setup, and analysis of TaqMan qRT-PCR experiments are discussed. Curr. Protoc. Microbiol. 15:1D.1.1-1D.1.13. © 2009 by John Wiley & Sons, Inc.

Keywords: quantitative reverse transcriptase-PCR (qRT-PCR); TaqMan; RNA; gene expression; Haemophilus influenzae

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: RNA Extraction from Haemophilus influenzae Cultures
  • Basic Protocol 2: RNA Cleanup and Assessment
  • Basic Protocol 3: TaqMan qRT-PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: RNA Extraction from Haemophilus influenzae Cultures

 Materials
  • BHI broth (see recipe)
  • Hemin (see recipe)
  • NAD stock solution (see recipe)
  • Plated cultures of various strains of H. influenzae (grown overnight on sBHI agar plates)
  • Supplemented brain heart infusion (sBHI) broth (see recipe)
  • RNase-free 10% SDS (Ambion), sterile
  • Acid phenol (see recipe)
  • Acid phenol:chloroform (Ambion)
  • Chloroform
  • 3 M sodium acetate, sterile and RNase-free (Ambion)
  • Isopropanol
  • 70% (v/v) ethanol (diluted in ultrapure water)
  • Sterile, RNase-free ultrapure water (Invitrogen)
  • Powder-free exam gloves
  • 125-ml culture flasks, sterile
  • 2-ml microcentrifuge tubes, sterile
  • Vortex
  • Disposable cuvettes
  • Spectrophotometer set to 600 nm
  • Shaking incubator set at 37°C
  • RNase-free 50-ml polypropylene conical tubes capable of centrifugation at 6000 × g, sterile
  • 80°C water bath
  • Ice water bath
  • Floor model centrifuge with a fixed-angle rotor capable of spinning 50-ml conical tubes
  • 25-ml disposable pipets, sterile
  • 1-ml and 100-µl micropipettors and RNase-free barrier tips
  • 1.5-ml RNase-free microcentrifuge tubes (Ambion), sterile
  • Microcentrifuge
  • Kimwipes, optional

Basic Protocol 2: RNA Cleanup and Assessment

 Materials
  • Dried RNA (see Basic Protocol 1)
  • Sterile, RNase-free ultrapure water (Invitrogen)
  • DNase I (New England Biolabs)
  • Super RNaseIN (Ambion)
  • RNeasy Mini Kit (Qiagen) containing:
    • Buffer RLT
    • RNease Mini spin column
    • Buffer RPE
  • 100% (v/v) ethanol, RNase-free
  • Ice
  • Agilent RNA 6000 Nano Kit
  • 1-ml, 200-µl, 20-µl, 10-µl micropipettors and RNase-free barrier tips
  • Vortex
  • Microcentrifuge
  • Microcentrifuge tube rack
  • 37°C incubator or heat block
  • 1.5-ml and 0.5-ml microcentrifuge tube, sterile and RNase-free
  • Nanodrop ND-1000 Spectrophotometer
  • Kimwipes
  • Agilent 2100 Bioanalyzer

Basic Protocol 3: TaqMan qRT-PCR

 Materials
  • RNA samples (see Basic Protocol 2)
  • RNase-free ultrapure water (Invitrogen), sterile
  • TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems) containing:
    • 2× RT-PCR mix
    • 40× RT Enzyme mix
  • Custom TaqMan Gene Expression Assays for endogenous control and target genes (Applied Biosystems)
  • Ice
  • RNase-free 0.5-ml microcentrifuge tubes (Ambion), sterile
  • Vortex
  • MicroAmp Fast Optical 96-Well Reaction Plates (0.1 ml; Applied Biosystems)
  • MicroAmp 96-Well Support Base (Applied Biosystems)
  • MicroAmp Optical Adhesive Film (Applied Biosystems)
  • Microcentrifuge
  • 8-channel multichannel pipettor
  • Benchtop centrifuge w/ 96-well plate supports
  • StepOne Plus Real-Time PCR Systems (Applied Biosystems)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 1D.1.1
    Schematic of the TaqMan assay. Assays consist of primers and a probe. The probe is constructed with a fluorescent dye (reporter, R) at the 5¢ end and a quencher (Q) at the 3¢ end. Degradation of the probe annealed to its specific target by the polymerase separates the reporter from the quencher, resulting in a net increase in fluorescence that can be measured after each cycle.

    View Image

Literature Cited

Literature Cited
    Fleischmann, R.D., Adams, M.D., White, O., Clay ton, R.A., Kirkness, E.F., Kerlavage, A.R., Bult, C.J., Tomb, J.F., Dougherty, B.A., Merrick, J.M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C., Gocayne, J.D., Scott, J., Shirley, R., Liu, L., Glodek, A., Kelley, J.M., Weidman, J.F., Phillips, C.A., Spriggs, T., Hedblom, E., Cotton, M.D., Utterback, T.R., Manna, M.C., Nguyen, D.T., Saudek, D.M., Brandon, R.C., Fine, L.D., Fritchman, J.L., Fuhrmann, J.L., Geoghagen, N.S.M., Gnehm, C.L., McDonald, L.A., Small, K.V., Fraser, C.M., Smith, H.O., and Venter, J.C. 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496-512.
    Harrison, A., Dyer, D.W., Gillaspy, A., Ray, W.C., Mungur, R., Carson, M.B., Zhong, H., Gip son, J., Gipson, M., Johnson, L.S., Lewis, L., Bakaletz, L.O., and Munson, R.S. Jr. 2005. Genomic sequence of an otitis media isolate of nontypeable Haemophilus influenzae: Comparative study with H. influenzae serotype d, strain KW20. J. Bacteriol. 187:4627-4636.
    Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M. 1996. Real time quantitative PCR. Gen. Res. 6:986-994.
    Kaplan, B., Wandstrat, T.L., and Cunningham, J.R. 1997. Overall cost in the treatment of otitis media. Pediatr. Infect. Dis. J. 16:S9-S11.
    Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–CT method. Methods 25:402-408.
    Murphy, T.F. and Apicella, M.A. 1987. Nontypable Haemophilus influenzae: A review of clinical aspects, surface antigens, and the human response to infection. Rev. Infect. Dis. 9:1-15.
    Smith-Vaughan, H.C., Sriprakash, K.S., Leach, A.J, Mathews, J.D., and Kemp, D.J. 1998. Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic. Infect. Immun. 66:3403-3409.
     
 
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