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Abstract

Introduction

Protocol

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Acknowledgements

Materials

References

Immunology and Infection

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Published: September 24th, 2021

DOI:

10.3791/62755

1Translational Medicine, Hospital for Sick Children, 2Department of Cell and Systems Biology, University of Toronto, 3Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 4Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Most chronic bacterial infections, such as pulmonary infections in cystic fibrosis (CF) patients and prosthesis related infections, are characterized by the growth of organisms within biofilms. Biofilms1 are communities of bacteria encased in a matrix composed primarily of polysaccharides2. Bacteria within biofilms can be slow growing, metabolically dormant, and in anaerobic, hypoxic conditions. Biofilms are more resistant to antibiotics due to factors such as decreased antibiotic penetration, increased expression of drug efflux pumps, and decreased cell division3. For these and other reasons, the....

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The Research Ethics Board (REB) is required for the collection and processing of sputum samples from human subjects. This study was approved by the Hospital for Sick Children (REB#1000019444). Research Ethics Board (REB) is required to collect and store sputum samples from human subjects. These studies were approved by the Hospital for Sick Children REB#1000058579.

1. Biofilm formation

  1. Grow Pseudomonas aeruginosa isolates obtained from the sputum samples of CF patients use.......

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The general overview of the method is shown in Figure 1. We previously used 8-well chamber slides to grow P. aeruginosa biofilms and expose them to antibiotics before then examining them via confocal microscopy at different time points12,13. This method can be used to extract total RNA directly from biofilms grown in this system in order to study gene expression changes post treatment. This protocol has been optimized for

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Total RNA is successfully extracted from 17 different bacterial biofilm samples in triplicate, yielding a total of 51 samples. The forty-nine RNA libraries are pooled and successfully sequenced. Overall, this validates our quality criteria with a 96 % success rate even though more than half the samples are considered to be low abundance and of sub-optimal quality34,35,36,37.

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Author contributions: P.W., Y.Y. and V.W were involved in conceptualizing the study. K.G., L.J., A.M. and P.W. optimized the lab protocols. Funding for K.G. was supported by the Student Work Placement Program subsidy through BioTalent Canada.

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NameCompanyCatalog NumberComments
Agilent 2100 BioanalyzerAgilentG2939BAAutomated electrophoresis of biomolecules
Agilent RNA 6000 pico kitAgilent5067-1513High sensitivity RNA electrophoresis chip to generate a RIN
DNA/RNA Lysis BufferZymo ResearchD7001-1-50A guanidinium thiocyanate and N-Lauroylsarcosine-based lysis buffer sold as part of a nucleic acid purification kit
DNA/RNA Prep BufferZymo ResearchD7010-2-10A guanidine HCl and ethanol buffer used for purification of DNA and RNA
DNA/RNA ShieldZymo ResearchR1100-50DNA and RNA preservation/protection reagent
DNA/RNA Wash BufferZymo ResearchD7010-3-6A salt and ethanol buffer used for purification of DNA and RNA
DNBSEQ G-400RSMGIG-400RSHigh throughput sequencer
MGIEasy RNA Directional Library Prep SetMGI1000006386Generate libraries for MGI high-throughput sequencing platforms from total RNA.
Mini-Beadbeater-96BioSpec1001A high energy, high throughput cell disrupter
NEBNext rRNA Depletion Kit (bacteria)New England BiolabsE7850XEfficient and specific depletion of bacterial rRNA (5S, 16S, 23S)
Nunc Lab-Tek II chamber slide systemThermo Fisher Scientific1545348-well chamber slide with removable wells
Qubit FluorometerThermo Fisher ScientificQ33238Fluorometer for DNA, RNA and proteins
Qubit RNA HS Assay KitThermo Fisher ScientificQ32852High sensitivity fluorometric assay to measure RNA concentration
Spin-Away FiltersZymo ResearchC1006-50-FSilica-based spin column primarily used to bind or remove genomic DNA
Sterile inoculation loops, 1 uLSarstedt86.1567.050Sterile, disposable inoculation loops for manipulation of microorganisms
ZR BashingBead Lysis tubesZymo ResearchS6003-502 mL tubes containing 0.1 and 0.5 mm bead lysis matrix for homogenizing biological samples
Zymo Spin IIICG ColumnsZymo ResearchC1006-50-GSilica-based spin column for purification of DNA and RNA
Zymo-Spin III-HRC FiltersZymo ResearchC1058-50Remove inhibitors such as polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc.
Zymobiomics DNA/RNA Miniprep kitZymo ResearchR2002DNA and RNA dual extraction kit
Zymobiomics HRC Prep solutionZymo ResearchD4300-7-30To be used with Zymo-Spin III-HRC Filters to remove PCR inhibitors

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