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DNA Isolation

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

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Related Experiment Video

Updated: Jul 31, 2025

Conjunctival Commensal Isolation and Identification in Mice

Conjunctival Commensal Isolation and Identification in Mice

Published on: May 1, 2021

DNA extraction protocol impacts ocular surface microbiome profile.

Heleen Delbeke^1,2, Ingele Casteels^1,2, Marie Joossens³

¹Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium.

Frontiers in Microbiology

|May 8, 2023

Summary

This summary is machine-generated.

Choosing the right DNA extraction protocol is crucial for accurate ocular microbiome analysis. This study provides a framework to compare methods, revealing significant differences in microbial composition and yield based on the protocol used.

Keywords:

16SrRNA sequencing DNA extraction protocol eye microbiome ocular surface microbiome sequencing results

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Area of Science:

Microbiology
Genomics
Bioinformatics

Background:

The ocular surface microbiome is increasingly studied using 16S ribosomal ribonucleic acid (rRNA) amplicon sequencing.
Lack of standardization in DNA extraction protocols complicates the comparison of published studies.

Purpose of the Study:

To establish a reference frame for interpreting and comparing ocular microbiome studies using different DNA extraction protocols.
To assess the quantitative and qualitative yield and reproducibility of various DNA extraction methods.

Main Methods:

Five commercial DNA extraction kits and two host DNA depletion kits were evaluated.
16S rRNA gene V3-V4 region sequencing was performed on ocular samples from seven volunteers.
Bioinformatic analysis included DADA2 for processing, SILVA for taxonomic assignment, and LEfSe for biomarker identification.

Main Results:

PowerSoil, RNeasy, and NucleoSpin kits yielded the highest DNA.
Significant differences in microbial composition were observed between extraction protocols, persisting after decontamination.
Eighteen bacterial genera were consistently detected across all non-pooled extraction kits.

Conclusions:

DNA extraction protocol significantly impacts ocular microbiome profiling.
A standardized reference frame is needed to interpret existing studies and guide future research.
Identifying protocol-specific contaminants and discriminative genera is essential for accurate analysis.