MARVICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v1 (protocols.io.bh82j9ye)

MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v2

10.17504/protocols.io.b2p8qdrw ◽

2021 ◽

Author(s):

Mo Li ◽

Gerardo Ramos-Mandujano

Keyword(s):

Extraction Method ◽

Magnetic Nanoparticle ◽

Rna Extraction ◽

Rna Isolation ◽

Emerging Pathogens ◽

Nucleic Acid Isolation ◽

Guanidinium Thiocyanate ◽

Biology Laboratory ◽

Alternative Solutions ◽

Basic Equipment

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

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MARVICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v2 (protocols.io.bik4kcyw)

protocols.io ◽

10.17504/protocols.io.bik4kcyw ◽

2020 ◽

Author(s):

Mo Li ◽

Gerardo Ramos

Keyword(s):

Extraction Method ◽

Magnetic Nanoparticle ◽

Rna Extraction

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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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Integrated Metabolomics and Transcriptomics Using an Optimised Dual Extraction Process to Study Human Brain Cancer Cells and Tissues

Metabolites ◽

10.3390/metabo11040240 ◽

2021 ◽

Vol 11 (4) ◽

pp. 240

Author(s):

Alison Woodward ◽

Alina Pandele ◽

Salah Abdelrazig ◽

Catherine A. Ortori ◽

Iqbal Khan ◽

...

Keyword(s):

Metabolic Pathways ◽

Extraction Method ◽

Limit Of Detection ◽

Rna Extraction ◽

Extraction Process ◽

Extraction Methods ◽

Serum Starvation ◽

Extraction Techniques ◽

Metabolic Genes ◽

Dual Extraction

The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme–metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite (sequential solvent addition and shaking) single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue.

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Detection of Schmallenberg Virus RNA in Bull Semen in Poland

Polish Journal of Veterinary Sciences ◽

10.1515/pjvs-2016-0083 ◽

2016 ◽

Vol 19 (3) ◽

pp. 655-657 ◽

Cited By ~ 6

Author(s):

J. Kęsik-Maliszewska ◽

M. Larska

Keyword(s):

Extraction Method ◽

Rna Extraction ◽

Viral Rna ◽

Schmallenberg Virus ◽

Rt Pcr ◽

Bovine Semen ◽

Bull Semen ◽

Virus Rna ◽

Virus Excretion ◽

Venereal Transmission

Abstract The detection of Schmallenberg virus (SBV) in the breeding bull semen raised the question of the possibility of venereal transmission of SBV which could result in cost-intensive restrictions in the trade of bovine semen. In order to evaluate the presence of SBV RNA in bovine semen, 131 bull semen samples from four locations in Poland collected between 2013 and 2015 were analysed by RT-PCR for viral RNA. SBV RNA was detected in 5.3% of the samples. The study has revealed that application of an appropriate RNA extraction method is crucial to detect virus excretion via semen.

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A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽

10.1007/s13205-020-02514-9 ◽

2020 ◽

Vol 10 (12) ◽

Author(s):

Siti Suriawati Badai ◽

Omar Abd Rasid ◽

Ghulam Kadir Ahmad Parveez ◽

Mat Yunus Abdul Masani

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Oil Palm ◽

Real Time Pcr ◽

Extraction Method ◽

Rna Extraction ◽

Quantitative Real Time Pcr

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Establishing an RNA extraction method from a small number of Demodex mites for transcriptome sequencing

Experimental Parasitology ◽

10.1016/j.exppara.2019.03.006 ◽

2019 ◽

Vol 200 ◽

pp. 67-72

Author(s):

Li Hu ◽

Yae Zhao ◽

Dongling Niu ◽

Rui Yang

Keyword(s):

Transcriptome Sequencing ◽

Extraction Method ◽

Rna Extraction ◽

Demodex Mites

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Rapid Virus Detection Procedure for Molecular Tracing of Shellfish Associated with Disease Outbreaks

Journal of Food Protection ◽

10.4315/0362-028x-70.4.967 ◽

2007 ◽

Vol 70 (4) ◽

pp. 967-974 ◽

Cited By ~ 25

Author(s):

ANA MARIA de RODA HUSMAN ◽

FROUKJE LODDER-VERSCHOOR ◽

HAROLD H. J. L. van den BERG ◽

FRANÇOISE S. LE GUYADER ◽

HILDE van PELT ◽

...

Keyword(s):

Extraction Method ◽

Rna Extraction ◽

Virus Detection ◽

Disease Outbreaks ◽

Extraction Methods ◽

Virus Rna ◽

Pathogenic Viruses ◽

Rna Extraction Methods ◽

Digestive Glands ◽

Outbreak Situation

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III–spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.

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