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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 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 protocol 2 )
RNase‐free ultrapure water (Invitrogen), sterile
TaqMan RNA‐to‐C_T 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)

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Figures

Figure 1.D0.1 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.

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Literature Cited

Literature Cited
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