scholarly journals Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

2021 ◽  
Author(s):  
Jesús Ortega-Pinazo ◽  
Pedro Jesús Serrano-Castro ◽  
Margarita Vida-Botella ◽  
Beatriz Martínez ◽  
María Jesús Pinto-Medel ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Rna Extraction ◽  
Extraction Process ◽  
Extraction Methods ◽  
Biological Matrices ◽  
Peripheral Blood Mononuclear ◽  
Experimental Procedure ◽  
Rna Quality ◽  
The Matrix ◽  
Rna Extraction Methods

Abstract Nucleic acids, RNA among them, are widely used in biomedicine. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher. In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. These data contribute to facilitate researchers in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure to guarantee both quality standards and its reproducibility.

scholarly journals Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

2021 ◽  
Author(s):  
Jesús Ortega-Pinazo ◽  
Pedro Jesús Serrano-Castro ◽  
Margarita Vida-Botella ◽  
Beatriz Martínez ◽  
María Jesús Pinto-Medel ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Rna Extraction ◽  
Extraction Process ◽  
Extraction Methods ◽  
Biological Matrices ◽  
Peripheral Blood Mononuclear ◽  
Experimental Procedure ◽  
The Matrix ◽  
Rna Extraction Methods ◽  
Selection Of

Abstract Nucleic acids, RNA among them, are widely used in biomedicine. Because of its susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher. In this study, we have compared seven different RNA extraction methods (automated, semi-automated, and manual) from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality, and functionality according to the method employed for RNA extraction and the matrix used. These data contribute to facilitate researchers in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure to guarantee both quality standards and its reproducibility.

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 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.

scholarly journals Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology

2016 ◽  
Vol 50 (2) ◽  
pp. 108-115 ◽  
Author(s):  
Alves Mônica Ghislaine Oliveira ◽  
Mario Pérez-Sayáns ◽  
Maria-Elena Padín-Iruegas ◽  
Maria Dolores Reboiras-López ◽  
José Manuel Suarez-Peñaranda ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Extraction Methods ◽  
Oral Cytology

scholarly journals Optimal RNA Extraction Methods and Development of Synthetic Clones for Seven Strawberry Viruses

2020 ◽  
Vol 26 (3) ◽  
pp. 170-178
Author(s):  
Sun-Jung Kwon ◽  
Ju-Yeon Yoon ◽  
In-Sook Cho ◽  
Bong-Nam Chung
Keyword(s):  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Extraction Methods

scholarly journals Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

2011 ◽  
Vol 77 (18) ◽  
pp. 6476-6485 ◽  
Author(s):  
Zhanbei Liang ◽  
Ann Keeley
Keyword(s):  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Direct Detection ◽  
Rna Extraction ◽  
Milk Powder ◽  
Extraction Methods ◽  
Content Type ◽  
Total Rna ◽  
Rna Extraction Methods

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

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.

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