scholarly journals Optimized DNA extraction and purification method for characterization of bacterial and fungal communities in lung tissue samples

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Vicente Pérez-Brocal ◽  
Fabien Magne ◽  
Susana Ruiz-Ruiz ◽  
Carolina A. Ponce ◽  
Rebeca Bustamante ◽  
...  
Keyword(s):  
Microbial Community ◽  
Dna Extraction ◽  
Lung Tissue ◽  
Dna Amount ◽  
Purification Method ◽  
Tissue Samples ◽  
Dna Yield ◽  
Microbial Profile ◽  
Extraction And Purification ◽  
Pre Treatment

Abstract Human lungs harbor a scarce microbial community, requiring to develop methods to enhance the recovery of nucleic acids from bacteria and fungi, leading to a more efficient analysis of the lung tissue microbiota. Here we describe five extraction protocols including pre-treatment, bead-beating and/or Phenol:Chloroform:Isoamyl alcohol steps, applied to lung tissue samples from autopsied individuals. The resulting total DNA yield and quality, bacterial and fungal DNA amount and the microbial community structure were analyzed by qPCR and Illumina sequencing of bacterial 16S rRNA and fungal ITS genes. Bioinformatic modeling revealed that a large part of microbiome from lung tissue is composed of microbial contaminants, although our controls clustered separately from biological samples. After removal of contaminant sequences, the effects of extraction protocols on the microbiota were assessed. The major differences among samples could be attributed to inter-individual variations rather than DNA extraction protocols. However, inclusion of the bead-beater and Phenol:Chloroform:Isoamyl alcohol steps resulted in changes in the relative abundance of some bacterial/fungal taxa. Furthermore, inclusion of a pre-treatment step increased microbial DNA concentration but not diversity and it may contribute to eliminate DNA fragments from dead microorganisms in lung tissue samples, making the microbial profile closer to the actual one.

Optimized DNA Extraction and Purification Method for Characterization of Bacterial and Fungal Communities in Lung Tissue Samples

2019 ◽  
Author(s):  
Vicente Pérez-Brocal ◽  
Fabien Magne ◽  
Susana Ruiz-Ruiz ◽  
Carolina A. Ponce ◽  
Rebeca Bustamante ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Lung Tissue ◽  
Fungal Communities ◽  
Purification Method ◽  
Tissue Samples ◽  
Extraction And Purification

scholarly journals A DNA extraction protocol for improved DNA yield from individual mosquitoes

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1314 ◽  
Author(s):  
Catelyn C. Nieman ◽  
Youki Yamasaki ◽  
Travis C. Collier ◽  
Yoosook Lee
Keyword(s):  
Next Generation Sequencing ◽  
Dna Extraction ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Single Individual ◽  
Dna Amount ◽  
Dna Yield ◽  
Extraction Protocol ◽  
Genome Representation ◽  
Generation Sequencing

Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.

scholarly journals New DNA Extraction Method for the Detection of Pneumocystis in Lung Tissue Samples of Colonized Individuals

OBM Genetics ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. 1-1
Author(s):  
Susana Ruiz-Ruiz ◽  
◽  
Carolina A Ponce ◽  
Nicole Pesantes ◽  
Rebeca Bustamante ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Lung Tissue ◽  
Extraction Method ◽  
Tissue Samples ◽  
Dna Extraction Method

scholarly journals Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0262355
Author(s):  
Elinor Shvartsman ◽  
Meika E. I. Richmond ◽  
John J. Schellenberg ◽  
Alana Lamont ◽  
Catia Perciani ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Female Genital Tract ◽  
Alpha Diversity ◽  
Sample Type ◽  
Microbial Composition ◽  
Dna Yield ◽  
Microbial Profiling ◽  
Pcr Product ◽  
Purification Methods ◽  
Pre Treatment

Background The microbiota of the lower female genital tract plays an important role in women’s health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. Methods DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. Results DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil

2015 ◽  
Vol 53 (11) ◽  
pp. 767-775 ◽  
Author(s):  
Mathur Nadarajan Kathiravan ◽  
Geun Ho Gim ◽  
Jaewon Ryu ◽  
Pyung Il Kim ◽  
Chul Won Lee ◽  
...  
Keyword(s):  
Microbial Community ◽  
Dna Extraction ◽  
Loess Soil ◽  
Extraction And Purification

scholarly journals Effects of Plant Tissue and DNA Purification Method on Randomly Amplified Polymorphic DNA-based Genetic Fingerprinting Analysis in Carrot

1999 ◽  
Vol 124 (1) ◽  
pp. 32-38 ◽  
Author(s):  
L.S. Boiteux ◽  
M.E.N. Fonseca ◽  
P.W. Simon
Keyword(s):  
Dna Extraction ◽  
Dna Cleavage ◽  
Extraction Method ◽  
Extraction Methods ◽  
The Other ◽  
Dna Purification ◽  
Genetic Fingerprinting ◽  
Purification Method ◽  
Fingerprinting Analysis ◽  
Dna Yield

Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting analysis using six tissues obtained from inbred carrot (Daucus carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DNA cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and purification methods for the total amount of DNA. An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when compared with the other six nonorganic extraction methods. Of the tissues examined, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fresh tissue); followed by seeds (54 ng·mg-1), fresh leaves (48 ng·mg-1), lyophilized leaves (40 ng·mg-1), calli (22 ng·mg-1), and tap roots (4 ng·mg-1). For most of the preparations, the DNA showed no traces of degradation. However, DNA preparations were not consistently accessible to HindIII cleavage in all tissue-extraction method combinations. Uncut DNA was observed chiefly in extractions from flowers and fresh leaves suggesting a tissue-specific adverse effect on restriction endonuclease activity. Differences in RAPD band (amplicon) intensity and number were observed across tissues and DNA extraction methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD-based fingerprinting yielding a consistently higher number of more intense amplicons when compared to the other tissues. In flowers and seeds, only DNA obtained with the CTAB extraction method could be amplified. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. Judicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for reliable RAPD-based DNA fingerprinting analysis in carrot. In addition, our studies allowed the identification of a better combination of procedures for use in routine manipulations of carrot DNA such as RFLP-RAPD-based cultivar fingerprinting, molecular mapping, screening of transgenic plants, construction of genomic libraries, and gene cloning.

Comparison of DNA Extraction and Purification Methods from Different Soils for Metagenomic Sequencing

2014 ◽  
Vol 955-959 ◽  
pp. 306-309 ◽  
Author(s):  
Lin Hui Wu ◽  
Jian Li Liu ◽  
Jing Zeng ◽  
Ji Zhao
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Metagenomic Sequencing ◽  
Soil Dna ◽  
Dna Yield ◽  
Extraction And Purification ◽  
Purification Methods ◽  
The Impact ◽  
Different Soils

There is an increased interest in the extraction of nucleic acids from various environmental samples, since only a minority of naturally occurring microbes can be cultured using standard techniques. Nucleic acids extraction and purification from soils are extremely challenging due to the low biomass, high organic contents and high variability of soil types. This has been regarded as one of the major difficulties that hamper the development of soil microbial ecology study. No commercial nucleic acids kits currently available are capable of preparing the DNAs without modifications. The cost can be very high for DNA extraction from extreme environmental soil samples, such as soils that have extreme high or low pHs. In this work, we developed and optimized soil DNA extraction and purification methods on different soils and compared the impact of three different DNA extraction protocols on DNA yield and purity. For the three different types of soil we used, direct extraction obtained the highest DNA recover rate, but required more cleanup steps. MoBio PowerSoil® DNA Isolation Kit yields less but do not require as many downstream cleaning steps. Both of the two methods obtained a more abundant microbial community than Meta-G-NomeTMDNA Isolation Kit.

In situ microprobe quantitation of inorganic particle burden in paraffin sections of lung tissue

1988 ◽  
Vol 46 ◽  
pp. 990-991
Author(s):  
Jerrold L. Abraham
Keyword(s):  
Lung Tissue ◽  
Quantitative Information ◽  
Particulate Material ◽  
Paraffin Sections ◽  
Tissue Samples ◽  
Qualitative And Quantitative ◽  
The Past ◽  
Quantitative Analyses ◽  
Data Result

Inorganic particulate material of diverse types is present in the ambient and occupational environment, and exposure to such materials is a well recognized cause of some lung disease. To investigate the interaction of inhaled inorganic particulates with the lung it is necessary to obtain quantitative information on the particulate burden of lung tissue in a wide variety of situations. The vast majority of diagnostic and experimental tissue samples (biopsies and autopsies) are fixed with formaldehyde solutions, dehydrated with organic solvents and embedded in paraffin wax. Over the past 16 years, I have attempted to obtain maximal analytical use of such tissue with minimal preparative steps. Unique diagnostic and research data result from both qualitative and quantitative analyses of sections. Most of the data has been related to inhaled inorganic particulates in lungs, but the basic methods are applicable to any tissues. The preparations are primarily designed for SEM use, but they are stable for storage and transport to other laboratories and several other instruments (e.g., for SIMS techniques).

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