Optimizing Protocols for High-Quality RNA Extraction from Blood and Liver Tissues of the Broad-Snouted Caiman

2021 ◽  
Vol 28 (4) ◽  
pp. 197-204
Author(s):  
Evelyn Cecilia López González ◽  
Lucía Magdalena Odetti ◽  
Gisela Laura Poletta ◽  
Nancy Denslow ◽  
Kevin J. Kroll ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Biological Processes ◽  
High Quality ◽  
Efficient Procedure ◽  
Physiological Conditions ◽  
Environmental Perturbation ◽  
Preservation Methods ◽  
Cell Tissue ◽  
And Storage ◽  
Liver Tissues

Transcriptomic information provides fundamental insights into biological processes and can be used to determine gene expression in cell, tissue, or organism under specific physiological conditions, or in response to any environmental perturbation. Extraction of high quality RNA is a challenging step mainly in non-traditional organisms, and protocols for preservation and isolation need to be adjusted in many cases. In the present work, we aimed to develop a protocol for preservation and isolation of high-quality and quantity of RNA from blood and liver tissues of Caiman latirostris. Three preservation methods were tested: 1) flash freezing (LN2) and storage at –80°C; 2) RNAlater® conservation with progressive cooling up to –80°C); 3) preservation in TRIzol® reagent, flash freezing in LN2 and storage at –80°C. Methods 1 and 2 were tested for liver, while 2 and 3 for blood. Our results showed that both preservation methods resulted in excellent outcomes for liver samples. For blood samples however, TRIzol® preservation was an efficient procedure for adequate RNA quality, quantity, and integrity, while conservation in RNAlater® solution was inadequate in both quality and quantity for an optimal RNA extraction. Appropriate protocols were established for each tissue and are being used now for transcriptomic studies in this sentinel organism.

scholarly journals Optimized protocol for the extraction of RNA and DNA from frozen whole blood sample stored in a single EDTA tube

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hirotaka Yamagata ◽  
Ayumi Kobayashi ◽  
Ryouichi Tsunedomi ◽  
Tomoe Seki ◽  
Masaaki Kobayashi ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ethylenediaminetetraacetic Acid ◽  
Rna Extraction ◽  
Basic Research ◽  
Rna Degradation ◽  
Disodium Salt ◽  
Water Bath ◽  
High Quality ◽  
Blood Samples ◽  
Rna And Dna

AbstractCryopreservation of whole blood is useful for DNA collection, and clinical and basic research. Blood samples in ethylenediaminetetraacetic acid disodium salt (EDTA) tubes stored at − 80 °C are suitable for DNA extraction, but not for high-quality RNA extraction. Herein, a new methodology for high-quality RNA extraction from human blood samples is described. Quickly thawing frozen whole blood on aluminum blocks at room temperature could minimize RNA degradation, and improve RNA yield and quality compared with thawing the samples in a 37 °C water bath. Furthermore, the use of the NucleoSpin RNA kit increased RNA yield by fivefold compared with the PAXgene Blood RNA Kit. Thawing blood samples on aluminum blocks significantly increased the DNA yield by ~ 20% compared with thawing in a 37 °C water bath or on ice. Moreover, by thawing on aluminum blocks and using the NucleoSpin RNA and QIAamp DNA Blood kits, the extraction of RNA and DNA of sufficient quality and quantity was achieved from frozen EDTA whole blood samples that were stored for up to 8.5 years. Thus, extracting RNA from frozen whole blood in EDTA tubes after long-term storage is feasible. These findings may help advance gene expression analysis, as well as biomarker research for various diseases.

scholarly journals Gene Expression Profiles at Moxibustioned Site (ST36): A Microarray Analysis

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Hai-Yan Yin ◽  
Yong Tang ◽  
Sheng-Feng Lu ◽  
Ling Luo ◽  
Jia-Ping Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microarray Analysis ◽  
Expression Profiles ◽  
Alternative Therapy ◽  
Gene Expression Profiles ◽  
Biological Processes ◽  
Physiological Conditions ◽  
Pathological Conditions ◽  
Molecular Events ◽  
Zusanli Acupoint

As a major alternative therapy in Traditional Chinese Medicine, it has been demonstrated that moxibustion could generate a series of molecular events in blood, spleen, and brain, and so forth. However, what would happen at the moxibustioned site remained unclear. To answer this question, we performed a microarray analysis with skin tissue taken from the moxibustioned site also Zusanli acupoint (ST36) where 15-minute moxibustion stimulation was administrated. The results exhibited 145 upregulated and 72 downregulated genes which responded immediately under physiological conditions, and 255 upregulated and 243 downregulated genes under pathological conditions. Interestingly, most of the pathways and biological processes of the differentially expressed genes (DEGs) under pathological conditions get involved in immunity, while those under physiological conditions are involved in metabolism.

scholarly journals High-quality total RNA isolation from melon (Cucumis melo L.) fruits rich in polysaccharides

2017 ◽  
Vol 38 (4) ◽  
pp. 2201 ◽  
Author(s):  
Gabrielle Silveira de Campos ◽  
Ricardo Antônio Ayub ◽  
Rafael Mazer Etto ◽  
Carolina Weigert Galvão ◽  
Marília Aparecida Stroka ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Sodium Perchlorate ◽  
Rna Isolation ◽  
World Market ◽  
Molecular Techniques ◽  
Guanidine Thiocyanate ◽  
High Quality ◽  
Total Rna ◽  
High Concentrations ◽  
Cucumis Melo L

Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil’s main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/?-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/?-mercaptoethanol; T5) SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

scholarly journals Rapid and Reliable Method of High-Quality RNA Extraction from Diverse Plants

2014 ◽  
Vol 05 (21) ◽  
pp. 3129-3139 ◽  
Author(s):  
Saroj Kumar Sah ◽  
Gurwinder Kaur ◽  
Amandeep Kaur
Keyword(s):  
Reliable Method ◽  
Rna Extraction ◽  
High Quality

Optimal drying and storage conditions for purple rice based on expected high quality

2017 ◽  
Vol 42 (2) ◽  
pp. e13502 ◽  
Author(s):  
Nittaya Junka ◽  
Chalermchai Wongs‐Aree ◽  
Chaiwat Rattanamechaiskul
Keyword(s):  
Storage Conditions ◽  
High Quality ◽  
And Storage

Isolation of high-quality total RNA from rumen anaerobic bacteria and fungi, and subsequent detection of glycoside hydrolases

2011 ◽  
Vol 57 (7) ◽  
pp. 590-598 ◽  
Author(s):  
Pan Wang ◽  
Meng Qi ◽  
Perry Barboza ◽  
Mary Beth Leigh ◽  
Emilio Ungerfeld ◽  
...  
Keyword(s):  
Gene Expression ◽  
Solid Phase ◽  
Large Subunit ◽  
Rna Extraction ◽  
Small Subunit ◽  
Glycoside Hydrolases ◽  
Small Subunit Rrna ◽  
High Quality ◽  
Major Barrier ◽  
Total Rna

The rumen is one of the most powerful fibrolytic fermentation systems known. Gene expression analyses, such as reverse transcription PCR (RT-PCR), microarrays, and metatranscriptomics, are techniques that could significantly expand our understanding of this ecosystem. The ability to isolate and stabilize representative RNA samples is critical to obtaining reliable results with these procedures. In this study, we successfully isolated high-quality total RNA from the solid phase of ruminal contents by using an improved RNA extraction method. This method is based on liquid nitrogen grinding of whole ruminal solids without microbial detachment and acid guanidinium – phenol – chloroform extraction combined with column purification. Yields of total RNA were as high as 150 µg per g of fresh ruminal content. The typical large subunit/small subunit rRNA ratio ranged from 1.8 to 2.0 with an RNA integrity number (Agilent Technologies) greater than 8.5. By eliminating the detachment step, the resulting RNA was more representative of the complete ecosystem. Our improved method removed a major barrier limiting analysis of rumen microbial function from a gene expression perspective. The polyA-tailed eukaryotic mRNAs obtained have successfully been applied to next-generation sequencing, and metatranscriptomic analysis of the solid fraction of rumen contents revealed abundant sequences related to rumen fungi.

TRENDS IN THE USE OF PACKAGING MATERIALS AND PACKAGING METHODS FOR PRODUCTS CONTAINING LABILE BIOLOGICALLY ACTIVE SUBSTANCES

2020 ◽  
Author(s):  
Е.П. ВИКТОРОВА ◽  
А.Д. АЧМИЗ ◽  
М.В. ЛУКЬЯНЕНКО ◽  
С.О. СЕМЕНИХИН
Keyword(s):  
Biologically Active Substances ◽  
Biologically Active ◽  
Packaging Materials ◽  
High Quality ◽  
Advantages And Disadvantages ◽  
Concentrate Polymer ◽  
Oxidative Processes ◽  
Livestock Products ◽  
And Storage ◽  
Active Substances

При производстве качественной пищевой продукции особое внимание необходимо уделять ее упаковыванию и хранению. Витаминно-минеральные концентраты, используемые для приготовления пищевых продуктов и получения качественной безопасной продукции животноводства, содержат комплекс лабильных биологически активных веществ, для сохранности которых необходимо минимизировать окислительные процессы. Это обусловливает повышенные требования к упаковке таких продуктов. С целью выбора упаковочного материала и способа упаковки витаминно-минерального концентрата проведен тематический обзор публикаций отечественных и зарубежных ученых. Рассмотрены тенденции в области применения упаковочных материалов, указаны преимущества и недостатки биополимеров. На основе проведенного анализа установлено, что для упаковывания витаминно-минерального концентрата целесообразно в качестве материала упаковки использовать полимерные пленки, обладающие высокой свето- и газонепроницаемостью, позволяющей снизить скорость протекания окислительных процессов и, следовательно, сократить потери содержащихся в продукте лабильных биологически активных веществ. В качестве способа упаковки можно рекомендовать применять вакуумирование, обеспечивающее отсутствие кислорода воздуха в упаковке. When producing high-quality food products, special attention should be paid to their packaging and storage. Vitamin and mineral concentrates used for food preparation and production of high-quality safe livestock products contain a complex of labile biologically active substances, for the safety of which it is necessary to minimize oxidative processes. This leads to increased requirements for the packaging of such products. In order to select the material and method of packaging of vitamin and mineral concentrates, a thematic review of publications of domestic and foreign scientists was conducted. Trends in the use of packaging materials are considered, advantages and disadvantages of biopolymers are indicated. It was found that for packaging vitamin and mineral concentrate, polymer pellicle with high light and gas tightness, which allows reducing the rate of oxidative processes and, consequently, reducing the loss of labile biologically active substances contained in the product, should be used as a packaging material. Vacuuming, which ensures the absence of oxygen in the air in the package, can be recommended as a method of packaging.

Polymer nanoparticles for drug delivery – synthetic vs. biopolymers?

BioResources ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. 2181-2183
Author(s):  
Martin Gericke ◽  
Thomas Heinze
Keyword(s):  
Quality Standards ◽  
Synthetic Polymer ◽  
Molecular Structures ◽  
Polymer Nanoparticles ◽  
Cell Type ◽  
High Quality ◽  
Medical Products ◽  
Physiological Conditions ◽  
Supramolecular Architectures ◽  
Specific Tissue

Nanoparticles have a great prospect for therapeutic applications. They can protect drugs under physiological conditions and act as a matrix for directed delivery of drugs, e.g., to a specific tissue or cell type. Polymer-based nanomaterials are considered as highly effective in this regard. Their properties can be tailored to meet specific demands for given therapeutic purposes. Considering the high-quality standards placed on medical products, the question arises: Which type of polymer material should be employed? One might select synthetic polymer compounds, which are highly diverse in terms of the molecular structures and supramolecular architectures that can be created, or biopolymers such as polysaccharides that are renowned for their native biocompatibility.

scholarly journals Emerging Roles and Potential Applications of Non-Coding RNAs in Glioblastoma

2020 ◽  
Vol 21 (7) ◽  
pp. 2611 ◽  
Author(s):  
Carlos DeOcesano-Pereira ◽  
Raquel A. C. Machado ◽  
Ana Marisa Chudzinski-Tavassi ◽  
Mari Cleide Sogayar
Keyword(s):  
Cancer Type ◽  
Biological Processes ◽  
Small Nucleolar Rnas ◽  
Physiological Conditions ◽  
Master Regulators ◽  
Potential Applications ◽  
Non Coding Rnas ◽  
Regulatory Rnas ◽  
Nucleolar Rnas ◽  
Substantial Effort

Non-coding RNAs (ncRNAs) comprise a diversity of RNA species, which do not have the potential to encode proteins. Non-coding RNAs include two classes of RNAs, namely: short regulatory ncRNAs and long non-coding RNAs (lncRNAs). The short regulatory RNAs, containing up to 200 nucleotides, include small RNAs, such as microRNAs (miRNA), short interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). The lncRNAs include long antisense RNAs and long intergenic RNAs (lincRNAs). Non-coding RNAs have been implicated as master regulators of several biological processes, their expression being strictly regulated under physiological conditions. In recent years, particularly in the last decade, substantial effort has been made to investigate the function of ncRNAs in several human diseases, including cancer. Glioblastoma is the most common and aggressive type of brain cancer in adults, with deregulated expression of small and long ncRNAs having been implicated in onset, progression, invasiveness, and recurrence of this tumor. The aim of this review is to guide the reader through important aspects of miRNA and lncRNA biology, focusing on the molecular mechanism associated with the progression of this highly malignant cancer type.

scholarly journals Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum

2019 ◽  
Vol 43 ◽  
Author(s):  
Rafael Novais de Miranda ◽  
Caroline Marcela da Silva ◽  
Antonio Carlos da Mota Porto ◽  
Welison Andrade Pereira
Keyword(s):  
Gene Expression ◽  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Rna Extraction ◽  
White Mold ◽  
Basic Requirement ◽  
High Quality ◽  
Commercial Kits ◽  
Made In

ABSTRACT The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative.

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