Oct 01, 2020

Public workspaceCTAB extraction of DNA and RNA of respiratory samples for microbial work

DOI
dx.doi.org/10.17504/protocols.io.bf28jqhw
  • 1National Heart and Lung Institute, Imperial College, London, UK, SW3 6LY
Leah Cuthbertson
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DOI: dx.doi.org/10.17504/protocols.io.bf28jqhw
Protocol Citation: Leah Cuthbertson, Michael Cox, Phillip James, Colin P Churchward, Bill Cookson, Miriam Moffatt 2020. CTAB extraction of DNA and RNA of respiratory samples for microbial work. protocols.io https://dx.doi.org/10.17504/protocols.io.bf28jqhw
Manuscript citation:
Cowman SA, James P, Wilson R, Cookson WOC, Moffatt MF, Loebinger MR (2018) Profiling mycobacterial communities in pulmonary nontuberculous mycobacterial disease. PloS one. https://doi.org/10.1371/journal. pone.0208018
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 06, 2020
Last Modified: October 01, 2020
Protocol Integer ID: 36672
Keywords: DNA, Extraction, respiratory, microbial, microbiome, metagenomics, Respiratory,
Abstract
This extraction method is modified from Griffiths et al 2000 Applied Environmental Microbiology 66(12): 5488–5491 and DeAngelis et al 2009 ISME Journal 3 pp 168-178.
CITATION
Griffiths RI, Whiteley AS, O'Donnell AG, Bailey MJ (2000). Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition.. Applied and environmental microbiology.

CITATION
DeAngelis KM, Brodie EL, DeSantis TZ, Andersen GL, Lindow SE, Firestone MK (2009). Selective progressive response of soil microbial community to wild oat roots.. The ISME journal.
LINK
https://doi.org/10.1038/ismej.2008.103
In comparison with DNA extraction kits, nucleic acid yields tend to be higher, though it is more laborious for larger numbers of samples.The extraction uses CTAB and Phenol:Chloroform:Isoamyl alcohol for lysis and the precipitation of nucleic acid is PEG based. An alternative to bead-beating is described, which should only be used for extracting DNA from cultured isolates. Bead-beating is avoided in this case in order to minimise shearing of DNA prior to genomic DNA sequencing.For all other samples use bead-beating is recommended.



This Protocol will cover the extraction of the following sample types;

Bacterial isolates
Upper respiratory tract swabs (nasal and throat swabs)
Upper respiratory tract lavage and aspirates (nasopharyngeal aspirate, endotracheal aspirate, nasal lavage, Mouth wash)
Saliva
Sputum (expectorated spontaneously produced, and induced sputum)
Upper and lower respiratory tract synthetic absorptive matrix (SAM) strips
Lower respiratory tract samples obtained via bronchoscopy - lung brushes, bronchoalveolar lavage and pleural fluid
Lung tissue – human or mouse
Lung biopsies
Cerebral spinal fluid (CSF)

This is a double extraction protocol, you are adding a second volume of Phenol:Chloroform:Isoamyl alcohol to each sample, precipitating two aqueous phases per sample and then recombining the resulting DNA pellets.This approach significantly increases the yield of DNA from samples and strains.
Materials
MATERIALS
ReagentPhenol:Chloroform:Isoamyl alcohol (25:24:1) Saturated with 10 mM Tris, pH 8.0
ReagentHexadecyltrimethylammonium bromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6269
ReagentEthanol (molecular biology grade, ≥99.8%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #51976-500ML-F
ReagentSodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
ReagentChloroform:Isoamyl alcohol 24:1Merck MilliporeSigma (Sigma-Aldrich)Catalog #C0549
Reagentlysing matrix E Catalog #116914050
ReagentPhase Lock Gel Separation tube Heavy QuantaBioVWR International (Avantor)Catalog #733-2478
ReagentFastPrep-24 HomogenizerMP BiomedicalsCatalog #116004500
ReagentLinear PolyacrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #56575-1ml
ReagentAluminum ammonium sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #402816
ReagentPolyethylene glycol 6000 Merck MilliporeSigma (Sigma-Aldrich)Catalog #8074911000
Reagent0.5ml Plain Skirted TubeStarLabCatalog #E1405-2142
STEP MATERIALS
Reagent0.5ml Plain Skirted TubeStarLabCatalog #E1405-2142
ReagentPhase Lock Gel Separation tube Heavy QuantaBioVWR International (Avantor)Catalog #733-2478

Protocol materials
ReagentPolyethylene glycol 6000 Merck MilliporeSigma (Sigma-Aldrich)Catalog #8074911000
ReagentSodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
Reagentlysing matrix E Catalog #116914050
ReagentLinear PolyacrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #56575-1ml
Reagent0.5ml Plain Skirted TubeStarLabCatalog #E1405-2142
Reagent0.5ml Plain Skirted TubeStarLabCatalog #E1405-2142
ReagentPhase Lock Gel Separation tube Heavy QuantaBioVWR International (Avantor)Catalog #733-2478
ReagentEthanol (molecular biology grade, ≥99.8%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #51976-500ML-F
ReagentChloroform:Isoamyl alcohol 24:1Merck MilliporeSigma (Sigma-Aldrich)Catalog #C0549
ReagentPhase Lock Gel Separation tube Heavy QuantaBioVWR International (Avantor)Catalog #733-2478
ReagentFastPrep-24 HomogenizerMP BiomedicalsCatalog #116004500
ReagentHexadecyltrimethylammonium bromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6269
ReagentAluminum ammonium sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #402816
ReagentPhenol:Chloroform:Isoamyl alcohol (25:24:1) Saturated with 10 mM Tris, pH 8.0
Reagent0.5ml Plain Skirted TubeStarLabCatalog #E1405-2142
ReagentPhase Lock Gel Separation tube Heavy QuantaBioVWR International (Avantor)Catalog #733-2478
Safety warnings

  • Informal training by a competent person before task can be undertaken unsupervised
  • Appropriate PPE must be worn at all time
  • Recommend doubling up nitrile gloves for handling phenol
  • Work conducted at Containment Level 2
  • Primary samples may be handled, but phenol and chloroform and to a lesser extent CTAB are the greater risks, so performwork in the fume cupboard, rather than the biological safety cabinet. Do not handle samples that are suspected to contain pathogens above ACDP category 2.

  • Ensure correct waste disposal procedures are used. Waste solvents should be collected in labelled glass waste.
  • The CTAB buffer cannot be disposed of down the sink as it is an environmental hazard. Collect waste solution as above.
  • Note, when pipetting solvents, pre-wet the pipette tip by very gently pipetting up and down.This saturates the headspace within the tip with the volatile solvent and prevents vapour pressure from causing the tip to drip excessively when pipetting.
  • Sealed buckets/rotors should be used for centrifugation steps
  • Sample spillages should be disinfected with 70% ethanol followed by cleaning with Surfanios (Biolab, 059840)
Ensure clear and appropriate labelling of all stored samples
Before start
Prepare reagents:

CTAB extraction buffer:
1Part: 10% (w/v) CTAB (hexadecyltrimethylammonium bromide) in 1M NaCl
1 part: 0.5 M phosphate buffer (pH 8.0) in 1 M NaCl

Prepare Concentration1 Molarity (M) NaCl in milliQ water.
Use the Concentration1 Molarity (M) NaCl solution you have prepared in place of water for preparing the 10 % CTAB stock solution.
Prepare the phosphate buffer at pH 8.0.
- Prepare Concentration1 Molarity (M) NaH2PO4 (Monobasic phosphate) and Concentration1 Molarity (M) Na2HPO4 (Dibasic phosphate) stocks using Concentration1 Molarity (M) NaCl in place of water.They may require gentle heating and stirring in order to dissolve.
- CombineAmount15.9 mL of monobasic phosphate with Amount284.1 mL of dibasic phosphate and make up to Amount600 mL with Concentration1 Molarity (M) NaCl to achieve pH 8.0
Combine the phosphate buffer and CTAB solution 1:1 to complete the CTAB extraction buffer
Sterilise by autoclaving

PEG/NaCl precipitation solution:
30 % (w/v) polyethylene glycol 6000 in Concentration1.6 Molarity (M) NaCl
Sterilise by autoclaving

0.1M Aluminum ammonium sulphate:
Concentration0.1 Molarity (M) Aluminum ammonium sulfate (AlNH4(SO4)2.12H20, Sigma 402816)
Filter-sterilize through 0.2mm filter



Before you start
Before you start
Add working stock of Phenol:Chloroform:Isoamyl (P:C:I (25:24:1)) alcohol to a Amount50 mL Falcon tubes in the fume cupboard.

Note
Use a 10ml pippette to remove required volume of P:C:I from below the the top buffered layer in the stock bottle.
*For a new bottle ensure the buffer has been added and allowed to settle prior to starting the extraction.

Add working stock of Chloroform:Isoamyl (C:I (24:1)) alcohol to a Amount50 mL Falcon tubes in the fume cupboard.

Pre-spin the 2 Phase-lock gel tubes (Amount2 mL Hard gel) per samples .Phase lock gel should be pelleted at the bottom of each tube (often on the sides when they arrive from manufacturer). Centrifuge at 16,000 x g forDuration00:05:00 .

ReagentPhase Lock Gel Separation tube Heavy QuantaBioVWR International (Avantor)Catalog #733-2478

Add Amount1 µL of linear polyacrylamide (LPA) to Amount2 mL eppendorfs, 2 per sample.This is a carrier and precipitates along with DNA increasing the yield. Unlike glycogen which can also be used it does not affect later sample use.

Extraction
Extraction
Add Amount50 µL aluminium ammonium sulfate to each Lysing Matrix E (LME) tube

Note
It is helpful to write the sample numbers on the lid and side of the LME tubes as the beadbeater can rub the numbers off.


Aseptically transfer sample to the LME tube and add 500ul of CTAB to the LME tube and incubate for Duration00:15:00 .



Note
Bacterial Isolates
a. Grow up bacterial isolates overnight in appropriate culture broth
b. Spin down Amount2 mL of broth for Duration00:10:00 at 16,000 x g
c. In a safety cabinet add 500ul of CTAB and resuspend the pellet. Incubate at room temperature for Duration00:15:00 .
d. Bacteria and CTAB can be stored frozen prior to extraction or extracted after Duration00:15:00 incubation.No additional CTAB is required.

Upper respiratory tract swabs (nasal and throat swabs)
a. Prepare 1 sterile spin baskets in a Amount1.5 mL trefflab tube per sample.
b. 1 pair of autoclaved scissors will be required per swab to transfer swab tips into bead beating tubes
c. Keep swabs on ice until transferred into LME tubes
d. Aseptically transfer swab tips into the LME tubes using sterile scissors.
e. Add Amount500 µL of CTAB to the LME tube and incubate for Duration00:15:00 .

Upper respiratory tract lavage and aspirates (nasopharyngeal aspirate, endotracheal aspirate, nasal lavage)
a. Spin a minimum volume of Amount2 mL , optimal volume 5mls of lavage fluid for Duration00:20:00 at full speed
b. Carefully resuspend pellet in Amount500 µL CTAB extraction buffer and transfer to LME tube.

NOTE:For saliva or mouth wash samples and sputum samples pre-alequoting using wide bore tips prior to storage is recommended.

Saliva or Mouth wash samples
a. Transfer a known volume of no more thanAmount300 µL into a LME tube.

Sputum (expectorated spontaneously produced, and induced sputum)
a. Transfer a known volume of no more than Amount300 µg or individual sputum plug, into a LME tube.

Upper and lower respiratory tract synthetic absorptive matrix (SAM) strips
a. 1 pair of autoclaved scissors will be required per SAM to transfer SAM into LME tubes
b. Keep SAMs on ice until transferred into LME tubes

Lung brushes
a. Keep lung brushes on ice until transferred to LME tubes

Bronchoalveolar lavage or Pleural fluid
a. Spin a minimum volume of Amount2 mL , optimal volume Amount5 mL of lavage fluid for Duration00:20:00 at full speed
b. Carefully resuspend pellet inAmount500 µL CTAB extraction buffer before transfering to LME tube.NO further CTAB is required

Lung tissue- Human or Mouse
a. Keep tissue on ice until transferred to LME tubes

Lung biopsies
a. Keep tissue on ice until transferred to LME tubes.


CSF
a.Maximise and standardise sample volume across the study.
b.Spin fluid for Duration00:20:00 at 16,000 x g
c.Carefully suspend pellet inAmount500 µL CTAB extraction buffer before transferring to LME tube.NO further CTAB is required


Safety information
Transfer of patient samples should be performed in a class 2 safety hood.



 Moving to a fume cupboard, carefully and immediately add Amount500 µL of P:C:I (25:24:1).  

Note
Pre-wet the pipette to avoid drips due to vapour pressure in tip.

Safety information
Perform this step in a fume hood

Safety information
Wear double gloves when handling P:C:I (25:24:1).Should a small spillage occur, outer gloves can then be disposed of without risk.


Safety information
 DO NOT leave tubes with P:C:I (25:24:1) for extended periods, it can degrade the plastic

Transfer to the bead-beater and beat using the pre-programmed CTAB setting for Duration00:01:00 . Return tubes to ice immediately after beating.


Note
CTAB Bead beating Program:

Speed: 5.5m/sec
Adapter: Quickprep
Time: 60 sec
Lysing Matrix: E
Quantity: 1ml
Cycles: 1
Pause time: 300sec

Note
Ensure the lids of the LME tubes are securely fastened with no beads in the seal, and that the tubes are labelled on the top and on the side as the

Centrifuge LME tubes at 16,000 x g for Duration00:05:00 .  

Transfer all liquid to phase lock gel tube and keep the LME tube on ice.


Note
Phase lock tubes should have been pre-spun. Phase lock gel should be pelleted at the bottom of each tube (often on the sides when they arrive from manufacturer). Centrifuge at 16,000 x g for Duration00:05:00


Centrifuge the phase lock tube at 16,000 x g forDuration00:05:00 at Temperature4 °C .

Note
Gel will form a barrier between the aqueous and P:C:I (25:24:1)/C:I (24:1) phases.

 Add 1 volume of C:I (24:1) to each phase lock gel tube, shake briefly to mix.  Centrifuge at 16,000 x g for Duration00:05:00 at Temperature4 °C .   

Note
If barrier does not form, extend centrifugation time. 


Second extraction; In the fume hood, add Amount50 µL of Aluminium ammonium sulphate, Amount500 µL of CTAB extraction buffer and Amount500 µL of P:C:I (25:24:1) to each bead beating tube.

 Repeat steps 8 to 13 then continue with the precipitation step below.
Precipitation
Precipitation
Transfer the aqueous phase from each tube to the pre-prepared eppendorfs with Amount1 µL of LPA in them. As the solvent is locked beneath the phase lock gel, you should be able to pour this. You may need 2 eppendorfs per sample to fit allow room for PEG.

Add 2 volumes of PEG/NaCl solution and mix well (solution is viscous). Leave overnight in the fridge at Temperature4 °C to precipitate.


Note
If you are in a hurry it can be left for 2 hours but this will reduce the yield.

Centrifuge all eppendorfs at 16,000 x g for Duration00:20:00 at Temperature4 °C .

Carefully aspirate the PEG/NaCl solution from the pellets.

Note
Pellets are usually large and translucent. They tend to be fairly uniform in size as the LPA has also precipitated.

Wash pellets with Amount500 µL ice-cold 70 % ethanol to remove any precipitated salts and centrifuge at 16,000 x g for Duration00:05:00 .

Repeat the wash twice with Amount200 µL ice-cold 70 % ethanol.

After briefly air drying the pellet for about 5 minutes, resuspend in Amount30 µL low EDTA TE and combine the resuspended total nucleic acid from each sample into a single tube (Amount60 µL total per sample). For sputum samples (or other high biomass samples) you may need to increase this volume to Amount50 µL per tube (Amount100 µL per sample).
Note
We do not recommend storing DNA in cryo tubes as you are unable to spin them down.
Recommended tubes:Tethered O ring, sterile tubes E1405-2142, star labs 

Reagent0.5ml Plain Skirted TubeVWR International (Avantor)Catalog #E1405-2142


This extract can be stored at Temperature-20 °C or Temperature-80 °C until required.

Citations
Griffiths RI, Whiteley AS, O'Donnell AG, Bailey MJ. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition.
DeAngelis KM, Brodie EL, DeSantis TZ, Andersen GL, Lindow SE, Firestone MK. Selective progressive response of soil microbial community to wild oat roots.
https://doi.org/10.1038/ismej.2008.103