Quick Emulsion PCR Extraction Protocol v1 (protocols.io.bcjpiumn)

Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.

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OPTIMATION OF DNA EXTRACTION PROTOCOL OF Elaeidobius kamerunicus

Jurnal Penelitian Kelapa Sawit ◽

10.22302/10.22302/iopri.jur.jpks.v24i2.10 ◽

2018 ◽

Vol 24 (2) ◽

pp. 77-86

Author(s):

Sri Wening ◽

Agus Eko Prasetyo ◽

Tjut Ahmad Perdana Rozziansha ◽

Agus Susanto

Keyword(s):

Dna Fingerprinting ◽

Dna Extraction ◽

Oil Palm ◽

Fruit Set ◽

Fragment Length Polymorphism ◽

Basic Knowledge ◽

Biological Study ◽

Palm Fruit ◽

Extraction Protocol ◽

Elaeidobius Kamerunicus

African pollination weevil (Elaeidobius kamerunicus Faust) has an important role in the productivity of Indonesian oil palm plantation. Up to now, there has not been a comprehensive biological study of the species at molecular level. The basic knowledge is very useful for exploitation of the weevil for effective oil palm fruit set development. This research aimed to obtain DNA extraction protocol of E. kamerunicus for DNA fingerprinting of the species. Results showed that using a DNA extraction kit,material disruption by using micro pestle resulted the highest quantity of DNA, while there were no significant differences of resulted DNA quantity among treatments using tissue lyser for material disruption. DNA extracted by using micro pestle or tissue lyser for material disruption is adequate for DNA fingerprinting using AFLP (Amplified Fragment Length Polymorphism) and sequencing techniques.

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Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽

10.3390/foods10081804 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1804

Author(s):

Daniel Plante ◽

Julio Alexander Bran Barrera ◽

Maude Lord ◽

Irène Iugovaz ◽

Neda Nasheri

Keyword(s):

Hepatitis A Virus ◽

Rna Extraction ◽

Complex Nature ◽

Murine Norovirus ◽

Rt Pcr ◽

Pcr Inhibitors ◽

Qrt Pcr ◽

Extraction Protocol ◽

Viral Particles ◽

Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

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Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

Virology Journal ◽

10.1186/s12985-021-01523-1 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Rachelle Bester ◽

Glynnis Cook ◽

Johannes H. J. Breytenbach ◽

Chanel Steyn ◽

Rochelle De Bruyn ◽

...

Keyword(s):

High Throughput ◽

Extraction Method ◽

Pathogen Detection ◽

High Throughput Sequencing ◽

Expression Profiles ◽

Rna Extraction ◽

Healthy Plant ◽

Ion Torrent ◽

Extraction Protocol ◽

Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

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An Acetic Acid-Based Extraction Protocol for the Recovery of U, Th and Pb from Calcium Carbonates for U-(Th)-Pb Geochronology

Geostandards and Geoanalytical Research ◽

10.1111/j.1751-908x.2013.00219.x ◽

2013 ◽

Vol 37 (3) ◽

pp. 261-275 ◽

Cited By ~ 10

Author(s):

Andrew J. Mason ◽

Gideon M. Henderson ◽

Anton Vaks

Keyword(s):

Acetic Acid ◽

Calcium Carbonates ◽

Extraction Protocol

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A multiparametric extraction method for Vn96-isolated plasma extracellular vesicles and cell-free DNA that enables multi-omic profiling

Scientific Reports ◽

10.1038/s41598-021-87526-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jeremy W. Roy ◽

Catherine A. Taylor ◽

Annie P. Beauregard ◽

Surendar R. Dhadi ◽

D. Craig Ayre ◽

...

Keyword(s):

Extracellular Vesicles ◽

Liquid Biopsy ◽

Small Rna Sequencing ◽

Isolation Method ◽

Protein Biomarkers ◽

Cell Free Dna ◽

Extraction Protocol ◽

Free Dna ◽

Rich Material ◽

Short Timeframe

AbstractExtracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and ‘omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.

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