Sampling of Intestinal Microbiota and Targeted Amplification of Bacterial 16S rRNA Genes for Microbial Ecologic Analysis

Maomeng Tong1, Jonathan P. Jacobs2, Ian H. McHardy1, Jonathan Braun1

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, California, 2 Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.41
DOI:  10.1002/0471142735.im0741s107
Online Posting Date:  November, 2014
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Abstract

Dysbiosis of host‐associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre‐processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology. © 2014 by John Wiley & Sons, Inc.

Keywords: intestinal microbiota; mucosal microbiota; lavage; Illumina sequencing; 16S rRNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Human Stool Sample Collection and Processing
  • Basic Protocol 2: Human Intestinal Tissue Biopsy Collection and Processing
  • Basic Protocol 3: Human Intestinal Mucosal Lavage Collection and Processing
  • Basic Protocol 4: Sampling of Mouse Intestinal Luminal and Mucosal Microbiota
  • Basic Protocol 5: Targeted PCR Amplification Using Barcoded Primers
  • Support Protocol 1: Computational Analysis of Illumina Sequencing Data
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Human Stool Sample Collection and Processing

  Materials
  • Dry ice
  • Liquid nitrogen
  • Bleach
  • 70% ethanol
  • Medium ambient IATA transport box (Therapak Corporation, cat. no. 23650)
  • 8‐oz. refrigerant pack (Therapak Corporation, cat. no. 56400)
  • Para‐Pak clean vials (Meridian Bioscience, cat. no. 900312)
  • Two biohazard specimen transport bags (Fisher Scientific, cat. no. 01‐800‐03)
  • Powder‐free nitrile exam gloves
  • Aluminum foil (Super Strength; VWR cat. no. 89093‐584)
  • Bench diapers
  • Mortar and pestle, 100 mm (Fisher Scientific, cat. no. S337621)
  • Metal spatulas, sterile
  • Cryovials, pre‐cooled
  • Class II, Type A2 biological safety cabinet
  • Biohazard container

Basic Protocol 2: Human Intestinal Tissue Biopsy Collection and Processing

  Materials
  • Phosphate‐buffered saline (PBS; )
  • Standard‐sized biopsy forceps
  • RNAlater TissueProtect tubes (Qiagen, cat. no. 76154)
  • Powder‐free nitrile exam gloves

Basic Protocol 3: Human Intestinal Mucosal Lavage Collection and Processing

  Materials
  • Sterile water for irrigation (Baxter, cat. no. 2F7114)
  • Slip‐tip disposable syringe, 60‐ml (BD, cat. no. 309654)
  • Mucous specimen trap, 40‐cc (Bard Medical, cat. no. 0035860)
  • Powder‐free nitrile exam gloves
  • 50‐ml high‐clarity polypropylene conical centrifuge tubes (BD, cat. no. 352098)

Basic Protocol 4: Sampling of Mouse Intestinal Luminal and Mucosal Microbiota

  Materials
  • Double distilled water
  • D10F medium: DMEM medium containing 10% fetal bovine serum (FBS)
  • D10F medium with 1 mM DTT
  • Extraction buffer (200 mM Tris, pH 8.0, 200 mM NaCl, 20 mM EDTA)
  • Dissection equipment, including forceps and razor blades
  • 1.5‐ml microcentrifuge tubes
  • 37°C orbital shaker
  • 70‐μm nylon mesh filter (Corning Life Sciences, cat. no. 352350)
  • Centrifuge (Beckman J‐6M with JS‐4.2 rotor)

Basic Protocol 5: Targeted PCR Amplification Using Barcoded Primers

  Materials
  • PowerSoil DNA isolation kit (MO BIO Laboratories, cat. no. 12888)
  • 96‐well plate of uniquely barcoded IL_806r primers (see Caporaso et al., )
  • ILHS_515f (see Caporaso et al., )
  • 10× PCR buffer (Sigma‐Aldrich, cat. no. D9307)
  • 10 mM dNTPs
  • JumpStart Taq DNA polymerase (Sigma‐Aldrich, cat. no. D9307)
  • DEPC‐treated water
  • QIAquick PCR purification kit (QIAGEN, cat. no. 28104)
  • Nanodrop spectrophotometer
  • Thermal cycler

Support Protocol 1: Computational Analysis of Illumina Sequencing Data

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Figures

Videos

Literature Cited

Literature Cited
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