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

Metabolites ◽  
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.

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

2007 ◽  
Vol 70 (4) ◽  
pp. 967-974 ◽  
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.

A comparative study of extraction techniques for maximum recovery of β-galactosidase from the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus

2020 ◽  
Vol 87 (1) ◽  
pp. 123-126 ◽  
Author(s):  
Rabin Gyawali ◽  
Ayowole Oyeniran ◽  
Tahl Zimmerman ◽  
Sulaiman O. Aljaloud ◽  
Albert Krastanov ◽  
...  
Keyword(s):  
Extraction Method ◽  
Polyacrylamide Gel Electrophoresis ◽  
Extraction Methods ◽  
Maximum Amount ◽  
Lactobacillus Delbrueckii ◽  
Extraction Techniques ◽  
Protein Determination ◽  
Assisted Extraction ◽  
Mechanical Methods ◽  
Mechanical Extraction

AbstractThe study reported in this research communication evaluates the chemical (solvents) and mechanical (sonication, bead-beater) extraction methods to determine the maximum recovery of β-galactosidase from L. bulgaricus spp. Among all extraction techniques, sonication-assisted extraction yielded the highest amounts of enzyme activity (between 1892–2156 Miller Units) in cell-free extract (supernatant). Interestingly, solvent extracted enzyme activities were found to be very low (between 83–153 Miller Units) in supernatant. SDS-polyacrylamide gel electrophoresis and the total protein determination showed that mechanical methods can completely lyse the cells. Our results thus demonstrated that the mechanical extraction method of sonication is the best one for recovering the maximum amount of lactase from L. bulgaricus strains.

scholarly journals Modified Spin Column-Based RNA Extraction Methods of Staphylococcus aureus using PureLink® RNA Mini Kit and Basic Laboratory Instrument

2021 ◽  
Vol 3 (2) ◽  
pp. 73-80
Author(s):  
Muhammad Taufiq Hidayat ◽  
Endry Nugroho Prasetyo
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Extraction ◽  
Extraction Process ◽  
Extraction Methods ◽  
Important Process ◽  
Sensitive Material ◽  
Bacterial Rna ◽  
Spin Column ◽  
Rna Extraction Methods ◽  
Transcriptomic Level

RNA extraction is an important process before gene expression assessment at the transcriptomic level. RNA is a sensitive material to environmental factors such as temperature and contaminants, so the RNA extraction process generally requires sophisticated and expensive laboratory instruments. In this study, we extract RNA from Staphylococcus aureus bacteria using the PureLink® RNA Mini Kit. The instruments used in this study are basic instruments such as a hand homogenizer and non-thermal centrifuge. The results of RNA extraction were visualized using agarose gel electrophoresis. These results indicate that bacterial RNA extraction can be performed using the PureLink® RNA Mini Kit even with inexpensive basic laboratory instruments.

scholarly journals First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan

2020 ◽  
Author(s):  
Eiji Haramoto ◽  
Bikash Malla ◽  
Ocean Thakali ◽  
Masaaki Kitajima
Keyword(s):  
River Water ◽  
Water Samples ◽  
Extraction Method ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Treatment Plant ◽  
Treated Wastewater ◽  
Wastewater Sample ◽  
River Water Samples ◽  
Wastewater Samples

ABSTRACTWastewater-based epidemiology is a powerful tool to understand the actual incidence of coronavirus disease 2019 (COVID-19) in a community because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can be shed in the feces of infected individuals regardless of their symptoms. The present study aimed to assess the presence of SARS-CoV-2 RNA in wastewater and river water in Yamanashi Prefecture, Japan, using four quantitative and two nested PCR assays. Influent and secondary-treated (before chlorination) wastewater samples and river water samples were collected five times from a wastewater treatment plant and three times from a river, respectively, between March 17 and May 7, 2020. The wastewater and river water samples (200–5,000 mL) were processed by using two different methods: the electronegative membrane-vortex (EMV) method and the membrane adsorption-direct RNA extraction method. Based on the observed concentrations of indigenous pepper mild mottle virus RNA, the EMV method was found superior to the membrane adsorption-direct RNA extraction method. SARS-CoV-2 RNA was successfully detected in one of five secondary-treated wastewater samples with a concentration of 2.4 × 103 copies/L by N_Sarbeco qPCR assay following the EMV method, whereas all the influent samples were tested negative for SARS-CoV-2 RNA. This result could be attributed to higher limit of detection for influent (4.0 × 103–8.2 × 104 copies/L) with a lower filtration volume (200 mL) compared to that for secondary-treated wastewater (1.4 × 102–2.5 × 103 copies/L) with a higher filtration volume of 5,000 mL. None of the river water samples tested positive for SARS-CoV-2 RNA. Comparison with the reported COVID-19 cases in Yamanashi Prefecture showed that SARS-CoV-2 RNA was detected in the secondary-treated wastewater sample when the cases peaked in the community. This is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in Japan.

scholarly journals Optimalisasi Teknik Ekstraksi dan Isolasi DNA Tanaman Suren (Toona Sureni Merr.) untuk Analisis Keragaman Genetik berdasarkan Random Amplified Polymorphic DNA (RAPD)

2012 ◽  
Vol 14 (1) ◽  
pp. 138 ◽  
Author(s):  
Muh Restu ◽  
Mukrimin Mukrimin ◽  
Gusmiaty Gusmiaty
Keyword(s):  
Genomic Dna ◽  
Extraction Method ◽  
Dna Isolation ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Amplification ◽  
Secondary Compounds ◽  
Extraction Methods ◽  
Extraction Techniques ◽  
High Quality ◽  
Optimum Extraction

The species of trees have different secondary compounds that need optimum extraction techniques. Appropriate extraction techniquesdetermine the quality and quantity of DNA produced. This research aims to found optimal of extraction methods and DNA isolation, thento created genome DNA in high quality and quantity, so that it can be using for genetic variation analyses in Suren (Toona sureni Merr.) byRandom Amplified Polymorphic DNA (RAPD). This study shows that DNA concentrates were 763.3 μg/ml, 180.0 μg/ml, 383.3 μg/ml, and436.7 μg/ml. While based on the results of PCR amplification using the primers OPD 03 shows that the four extraction methods used, the extraction method of number 3 has been able to produce genomic DNA with better quality and more number of bands, although the quantityis lower.

scholarly journals Evaluation of the efficacy of LAMP-based SARS-CoV-2 detection with simple RNA extraction from nasopharyngeal swabs: A prospective observational study

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260732
Author(s):  
Masaki Karino ◽  
Mizuki Harada ◽  
Chihiro Yamada ◽  
Kyoko Fukuoka ◽  
Megumi Sugo ◽  
...  
Keyword(s):  
Extraction Method ◽  
Prospective Observational Study ◽  
Medical Center ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Viral Rna ◽  
Nasopharyngeal Swab ◽  
Measurement Principle ◽  
Comparison Of The Results ◽  
Reliable Testing

The Loopamp SARS-CoV-2 Detection Kit is used for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loop-mediated isothermal amplification (LAMP) is based on a measurement principle that can be used with a relatively simple device. Detection using this kit requires viral RNA extraction from samples with the QIAGEN QIAamp Viral Mini Kit (QIAGEN extraction) or the Loopamp Viral RNA Extraction Kit (Eiken extraction), which are recommended by the manufacturer. However, the efficacy of LAMP-based SARS-CoV-2 detection using these extraction methods has not been compared. In this study, we aimed to compare the results of genome extraction and detection from nasopharyngeal swab samples using the QIAGEN and Eiken extraction kits. The present study involved patients who presented to the Rinku General Medical Center with suspected COVID-19 (25 positive and 26 negative cases). A comparison of the results obtained using each extraction method with those obtained via PCR showed that the positive, negative, and overall concordance rates between QIAGEN extraction and PCR were 96.0% (24/25 samples), 100% (26/26), and 98.0% (50/51; κ = 0.96, 95% CI = 0.69–1.00), respectively. Results with Eiken extraction were also favorable, with positive, negative, and overall concordance rates of 88.0% (22/25), 100% (26/26), and 94.1% (48/51; κ = 0.88, 95% CI = 0.61–1.00), respectively. Favorable results were obtained using both QIAGEN and Eiken extraction kits. Since Eiken extraction can be completed in a few minutes, it enables prompt and reliable testing for SARS-CoV-2 detection.

scholarly journals Development and validation of portable, field-deployable Ebola virus point-of-encounter diagnostic assay for wildlife surveillance

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Dania M. Figueroa ◽  
Eeva Kuisma ◽  
M. Jeremiah Matson ◽  
Alain U. Ondzie ◽  
Trent Bushmaker ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Extraction Method ◽  
Ebola Virus ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Cold Chain ◽  
Standard Laboratory ◽  
Spin Column ◽  
Republic Of The Congo ◽  
Human Primate

Abstract Early detection of Ebola virus spillover into wildlife is crucial for rapid response. We developed and validated a portable, cold-chain independent Ebola virus RT-qPCR assay. Methods The field syringe-based RNA extraction method was compared with a conventional laboratory-based spin-column RNA extraction method. Next, the qPCR efficiency and limit of detection of the assay was compared to standard laboratory-based reagents and equipment. The specificity of the assay was confirmed by testing against multiple Zaire Ebolavirus (EBOV) variants and other ebolavirus species. Lastly, swabs from an EBOV-infected non-human primate carcass, stored at environmental conditions mimicking central and west Africa, were analyzed to mimic in field conditions. Results The syringe-based RNA extraction method performed comparably to a standard laboratory spin-column-based method. The developed assay was comparable in sensitivity and specificity to standard laboratory-based diagnostic assays. The assay specifically detected EBOV and not any of the other tested ebolavirus species, including Reston ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, and Tai Forrest ebolavirus. Notably, the assays limit of detection for EBOV isolates were all below 4 genome copies/μL. The assay was able to detect EBOV in oral, nasal, thoracic cavity, and conjunctiva swabs obtained from an infected non-human primate. Conclusion We developed a field-based Ebolavirus assay which is comparable in sensitivity and specificity to laboratory-based assays. Currently, the assay is being incorporated into wildlife carcass surveillance in the Republic of the Congo and is being adapted for other infectious disease agents.

scholarly journals Application of Green Extraction Techniques for Natural Additives Production

2021 ◽  
Author(s):  
Anxo Carreira-Casais ◽  
Catarina Lourenço-Lopes ◽  
Paz Otero ◽  
María Carpena ◽  
Antia Gonzalez Pereira ◽  
...  
Keyword(s):  
Raw Materials ◽  
Extraction Process ◽  
Extraction Methods ◽  
World Market ◽  
Extraction Techniques ◽  
Chemical Additives ◽  
Green Extraction ◽  
Market Needs ◽  
Natural Foods ◽  
Natural Additives

During the last decades, consumers have increased the demand for healthier natural foods with lower presence of chemical additives. One reason of this choice is the controversy about chemical additives possible adverse effects. To fulfill market needs, different techniques have been developed to extract compounds from various raw materials to produce natural additives with different properties (preservatives, emulsifiers, or colorants) and bioactivities. In addition, the growing concern about the effects of climate change has led the development of more sustainable techniques to carry out the extraction. The use of new alternative nonconventional, emerging, or green extraction methodologies has gained considerable attention during the last decade. These novel techniques have been applied to minimize any negative changes in the nutritional, physicochemical or sensory properties of the natural source, while at the same time reducing the environmental impact of the process and gaining competitiveness of the world market. For this purpose, new green extraction methods have been proposed and optimized for the reduction of the consumption of raw materials, solvents, and energy. In this chapter, a revision of different types of green extraction techniques is compiled together with the main factor that can affect extraction-process feasibility and the main challenges and future trends for their development.

scholarly journals Study on the Effect of Different Extraction Methods in Four Pharmacopoeia on the Content and Structural Transformation of Ginsenoside and the Optimization of Extraction Process

2021 ◽  
Author(s):  
Kai Sun ◽  
Hua Jiang ◽  
Pingya Li ◽  
Lei Xu ◽  
Yaling Deng ◽  
...  
Keyword(s):  
United States ◽  
Structural Transformation ◽  
Extraction Method ◽  
The United States ◽  
Extraction Process ◽  
Extraction Methods ◽  
Detection Methods ◽  
Content Determination ◽  
Q Exactive

Abstract Background: In recent years, ginseng products are widely used in various fields. More and more people pay attention to the extraction methods and quality evaluation of ginseng. At present, China, the United States, Europe, Japan and Korea have the quality standards and content determination methods of ginseng. However, due to the different treatment methods adopted before the determination of ginseng samples, the content limits of the index components, such as ginsenoside Rb1, Rg1 and Re are also different. There have been literature analyzed the similarities and differences of ginseng content detection methods in pharmacopoeias of different countries, but the comparison of the effects of different methods on ginsenoside content and structural transformation has not been reported.Methods: In this paper, ginsenosides in ginseng were extracted according to four national Pharmacopoeia, and analyzed quantitatively and qualitatively by UPLC-Q-Exactive-MS and HPLC-UV. Finally, a simple and feasible extraction method was optimized by response surface method. Results: Twelve kinds of ginsenosides in ginseng were quantitatively analyzed by using the methods of four national pharmacopoeia. Among them, the contents of Rg1, Re and Rd were high, and they were the highest by using unheated J/KP (Japan/Korea Pharmacopoeia) method. Ten kinds of ginsenosides were determined by heated CP (China Pharmacopoeia), USP (the United States Pharmacopoeia) and EP (European Pharmacopoeia) method, and seven kinds of ginsenosides were determined by unheated J/KP method. In the following UPLC-Q-Exactive-MS study, 34, 36, 21 and 19 ginsenosides were identified by CP, USP, EP and J/KP method, respectively. In the optimization of ginsenoside extraction process, an efficient extraction method was selected from the solvent, extraction time, solid-liquid ratio and other factors. In conclusion, through the qualitative and quantitative comparison of CP, USP and EP samples after heating, it can be seen that ginsenoside heating will increase the content of rare saponins, and the heating time is directly proportional to the content of rare saponins. Conclusion: The pretreatment method has a significant effect on the content determination of ginseng. The analysis of the preparation method and process optimization of the four Pharmacopoeia can provide important reference for the revision of ginseng standard.

scholarly journals PERBANDINGAN METODE RENDERING BASAH DAN EKSTRAKSI PELARUT N-HEKSANA TERHADAP KARAKTERISTIK KIMIA MINYAK IKAN SELAR (Selaroides leptolepis)

2020 ◽  
Vol 5 (1) ◽  
pp. 21-25
Author(s):  
Nurfadilah
Keyword(s):  
Solvent Extraction ◽  
Fish Oil ◽  
Extraction Method ◽  
Unsaturated Fatty Acids ◽  
Human Growth ◽  
Extraction Process ◽  
Extraction Methods ◽  
Acid Number ◽  
Solvent Extraction Method ◽  
Long Chain

Fish oil contains nutrients that are good for human growth because generally it contains long-chain unsaturated fatty acids that have double bonds, namely eicosapenta-enoate (EPA), and docose-hexaenoate (DHA). Fish Oil is obtained from the extraction process, namely the process of separating fish oil from the meat. This study aims to determine the comparison of wet rendering methods and n-hexane solvent extraction on the chemical characteristics of selar fish oil (selaroides leptolepis). The method used in this research is descriptive experimental method by comparing the 2 extraction methods that are treated in this study. The extraction method used was the Wet Rendring Extraction Method and N-Hexane Solvent. The extracted fish oil was then characterized by the acid number, saponation number and peroxide number. The saponification number using the wet rendering extraction method (20.63 mg KOH / g) is relatively the same as the solvent extraction method (20.53). The peroxide value in the wet rendering extraction method (29.59 mek / kg) was higher than the n-hexane solvent extraction method (24.77 mek / kg). The acid number in the wet rendering extraction method (70.2 mg KOH / gr) was higher than the solvent extraction method (23.74 mg KOH / gr). Selar fish oil extracted using wet rendering method and n-hexane solvent has low quality

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