Example of Real‐Time Quantitative Reverse Transcription–PCR (Q‐RT‐PCR) Analysis of Bacterial Gene Expression during Mammalian Infection: Borrelia burgdorferi in Mouse Tissues

Jennifer C. Miller1

1 University of Kentucky College of Medicine, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1D.3
DOI:  10.1002/9780471729259.mc01d03s00
Online Posting Date:  October, 2005
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Abstract

This unit provides a chronological in‐depth description of all protocols needed for quantitative reverse transcription–PCR (Q‐RT‐PCR) analysis of Borrelia burgdorferi gene expression within infected mouse tissues. Specifically, this unit discusses the extraction of RNA from infected mouse tissues, removal of contaminating genomic DNA from the purified RNA, preparation of cDNA and genomic DNA standards, LightCycler‐based PCR, and a relative quantification analysis of the cDNA. Q‐RT‐PCR as a highly relevant and powerful tool used to detect gene expression by bacteria within mammalian host tissues. It is also an invaluable technique used to measure and assess the small differences in expression that can exist among a set of genes, whereas this type of analysis is not feasible using less‐sensitive techniques such as indirect immunofluorescence analysis or qualitative RT‐PCR. Although the protocols described herein are tailored for B. burgdorferi, they are broadly applicable to other microbes.

Keywords: quantitative RT‐PCR; mouse infection; Borrelia burgdorferi; gene expression; RNA; cDNA; LightCycler

     
 
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Table of Contents

  • Basic Protocol 1: Extraction of RNA from B. burgdorferi‐Infected Mouse Tissues
  • Basic Protocol 2: Quality Control: Removal of Contaminating Genomic DNA and Assessment of RNA Quality and Yield
  • Support Protocol 1: Spectrophotometric Determination of RNA Concentration
  • Basic Protocol 3: Quantitative RT‐PCR and Analysis
  • Support Protocol 2: Preparation of Genomic DNA Standards
  • Basic Protocol 4: Verification of Specificity of Lightcycler‐Based PCR Reactions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extraction of RNA from B. burgdorferi‐Infected Mouse Tissues

  Materials
  • RNase‐Away (Fisher Scientific)
  • Dry ice
  • Trizol reagent, stored at 4°C
  • RNAsecure resuspension solution (Ambion), stored at −20°C
  • Cryotubes containing B. burgdorferi–infected tissue, −80°C
  • Sterile, DEPC‐treated, double‐distilled water ( appendix 2A)
  • 5% (w/v) SDS (prepared in sterile, DEPC‐treated water)
  • 1‐Bromo‐3‐chloropane
  • 2‐ Propanol (isopropanol)
  • 75% ethanol (diluted in sterile, DEPC‐treated water)
  • Powder‐free exam gloves
  • Microcentrifuge tube racks
  • 1‐ml, 200‐µl, and 20‐µl micropipettors and corresponding RNase‐free barrier tips
  • 4 9/16–in. (∼11.59‐cm) blunt forceps
  • Ceramic crucible and pestle
  • Variable speed digital Dremel homogenizer (Fisher Scientific)
  • Ice buckets with lids
  • Aluminum foil cut into ∼2 × 2–in. (∼5 × 5–cm) squares
  • Sterile 12 × 75–mm, 5‐ml polystyrene round‐bottom conical tubes
  • Sterile, RNase‐free 5‐ml conical tubes (Ambion)
  • Sterile, RNase‐free 1.5‐ml microcentrifuge tubes (Ambion)
  • 60°C heating block
  • 50‐ml polypropylene conical tubes, sterile
  • Refrigerated microcentrifuge, 4°C
  • Freezer boxes

Basic Protocol 2: Quality Control: Removal of Contaminating Genomic DNA and Assessment of RNA Quality and Yield

  Materials
  • RNase‐Away (Fisher Scientific)
  • RNA extracted from B. burgdorferi–infected tissues, –80°C
  • 10× Turbo DNA‐free buffer (Turbo DNA‐free kit) treated with RNAsecure (Ambion)
  • 25× RNAsecure solution (Ambion), stored at −20°C
  • DNase inactivation reagent (Turbo DNA‐free kit) treated with RNAsecure (Ambion)
  • 10 U/µl RNase‐free DNase I (Roche Applied Science)
  • Agarose
  • 1× TBE: dilute from 5× TBE stock ( appendix 2A; also see Sambrook et al., ) in sterile, DEPC‐treated water ( appendix 2A) and treat with RNAsecure
  • Ethidium bromide staining solution ( appendix 2A)
  • 5× DNA loading dye (prepared in sterile, DEPC‐treated water and treated with RNAsecure; see Sambrook et al., )
  • 100‐bp DNA ladder (New England Biolabs)
  • Takara 10× dNTPs (Fisher Scientific)
  • Takara 10× PCR buffer (Fisher Scientific)
  • 30 µM B. burgdorferi Fla1 oligonucleotide primer (Miller et al., )
  • 30 µM B. burgdorferi Fla2 oligonucleotide primer (Miller et al., )
  • Takara Taq polymerase (Fisher Scientific)
  • 10‐µl micropipettor and barrier tips
  • 37° and 60°C heating blocks
  • Sterile, RNase‐free 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Agarose gel casting apparatus, gel trays, and combs
  • Agarose gel electrophoresis gel box
  • Electrophoresis power supply
  • UV light box
  • RNase‐free, sterile, thin‐walled PCR tubes
  • Thermal cycler

Support Protocol 1: Spectrophotometric Determination of RNA Concentration

  Materials
  • RNAsecure resuspension solution (Ambion), stored at −20°C
  • DNase I–treated RNA sample (see protocol 2)
  • RNase‐free 1.5‐ml microcentrifuge tubes
  • Spectrophotometer
  • Cuvettes

Basic Protocol 3: Quantitative RT‐PCR and Analysis

  Materials
  • RNase‐Away (Fisher Scientific)
  • DNA‐free RNA sample
  • Sterile, DEPC‐treated water ( appendix 2A)
  • DNA‐Away (Fisher Scientific)
  • First‐strand cDNA synthesis kit for RT‐PCR (AMV; Roche) containing the following:
    • 10× reaction buffer
    • 25 mM MgCl 2
    • 40 mM (10 mM each) dNTPs
    • 0.04 A 260 U/µl random primer p(dN)6
    • 50 U/µl RNase‐inhibitor
    • AMV reverse transcriptase
  • Enzyme diluent (Idaho Technology; also see recipe for SED in appendix 2A)
  • 10× dNTPs containing 2 mM each dNTP (Idaho Technology; also see appendix 2A)
  • 10× PCR buffer containing 30 mM MgCl 2 (Idaho Technology; also see appendix 2A)
  • 20 µM each of housekeeping gene primers Fla1 and Fla2 (Miller et al., ) or 20 µM of each oligonucleotide primer used to amplify gene of interest
  • Platinum Taq polymerase (Invitrogen)
  • Sterile TE buffer (made with sterile, DEPC‐treated water; appendix 2A)
  • 10,000× SYBR green (Molecular probes) diluted 1000× in TE (final concentration 10×) in amber‐colored, sterile, siliconized tubes
  • Genomic DNA standards (see protocol 5)
  • 2‐, 10‐, 20‐, and 200‐µl, and 1‐ml micropipettors and RNase‐free, DNA‐free barrier pipet tips
  • Clear, sterile, RNase‐free, DNA‐free, siliconized 0.5‐ml flat‐top microcentrifuge tubes (Fisher Scientific)
  • 42° and 99°C heating blocks
  • LightCycler centrifuge adapters in an aluminum cooling block (Roche), 4°C
  • 20‐µl LightCycler capillary tubes (Roche)
  • LightCycler thermal cycler with carousel (Roche)
  • Computer equipped with LightCycler software v. 3.5.3 (Roche)
  • Color printer

Support Protocol 2: Preparation of Genomic DNA Standards

  Materials
  • DNA‐easy tissue kit (Qiagen)
  • 5‐ml mid‐logarithmic phase culture of B. burgdorferi strain of interest (unit 12.1)
  • 0.7% agarose gel
  • RNase‐free TE buffer ( appendix 2A)
  • Spectrophotometer
  • Gel electrophoresis equipment
  • Sterile, RNase‐free, DNA‐free siliconized 0.5‐ml microcentrifuge tubes

Basic Protocol 4: Verification of Specificity of Lightcycler‐Based PCR Reactions

  Materials
  • Capillary tubes containing the LightCycler amplicons of interest ( protocol 2)
  • 1.5% agarose gel containing ethidium bromide
  • 5× loading dye
  • 100‐bp DNA ladder
  • 1.5‐ml microcentrifuge tubes
  • UV lightbox
  • Microcon‐100 microconcentrator filter (Ambion)
  • Spectrophotometer
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Figures

Videos

Literature Cited

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   Caimano, M.J., Eggers, C.H., Hazlett, K.R.O., and Radolf, J.A. 2004. RpoS is not central to the general stress response in Borrelia burgdorferi but does control expression of one or more essential virulence determinants. Infect. Immun. 72:6433‐6445.
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Key References
   Gilmore et al., 2001. See above.
  Gilmore and colleagues were the first investigators to utilize Q‐RT‐PCR to analyze B. burgdorferi gene expression.
   Miller et al., 2005. See above.
  This report utilizes all the protocol steps outlined in this unit to examine Borrelia burgdorferi gene expression within tissues of chronically infected non‐human primates.
Internet Resources
   http://www.Roche‐applied‐science.com/lightcycler‐online
  Excellent resource offering detailed technical notes on the LightCycler and its applications. The LightCycler Operator's Manual Version 3.5 is also available for downloading free of charge at this site.
   http://www.IDT.com
  Free Website offers online tools for the analysis of oligonucleotide primer sequences.
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