A Modified Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines

2016 ◽  
pp. 27-33 ◽  
Author(s):  
Júlio César de Lima ◽  
Thanise Nogueira Füller ◽  
Fernanda de Costa ◽  
Kelly C. S. Rodrigues-Corrêa ◽  
Arthur G. Fett-Neto
Keyword(s):  
Rna Extraction ◽  
High Quality

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.

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 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

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.

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.

scholarly journals A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Kucharski ◽  
Jaishree Tripathi ◽  
Sourav Nayak ◽  
Lei Zhu ◽  
Grennady Wirjanata ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Rna Extraction ◽  
Transcriptome Data ◽  
High Quality ◽  
Diverse Range ◽  
Human Haemoglobin ◽  
Infected Blood ◽  
Blood Samples ◽  
Total Rna

Abstract Background Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. Results The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest  2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to  date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. Conclusions Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

scholarly journals Evaluation of different RNA extraction methods for high-quality total RNA and mRNA from Erwinia amylovora in planta

2016 ◽  
Vol 146 (4) ◽  
pp. 893-899 ◽  
Author(s):  
Monika Kałużna ◽  
Anita Kuras ◽  
Artur Mikiciński ◽  
Joanna Puławska
Keyword(s):  
Erwinia Amylovora ◽  
Rna Extraction ◽  
Extraction Methods ◽  
In Planta ◽  
High Quality ◽  
Total Rna ◽  
Rna Extraction Methods

scholarly journals Comparative assessment of four RNA extraction methods and modification to obtain high-quality RNA from Parthenium hysterophorus leaf

3 Biotech ◽  
2017 ◽  
Vol 7 (6) ◽  
Author(s):  
Javed Ahmad ◽  
M. Affan Baig ◽  
Arlene A. Ali ◽  
Asma Al-Huqail ◽  
M. M. Ibrahim ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Extraction Methods ◽  
Comparative Assessment ◽  
Parthenium Hysterophorus ◽  
High Quality ◽  
Rna Extraction Methods

High quality RNA from hydroponically grown grapevine roots suitable for gene expression studies

2017 ◽  
Vol 42 (4) ◽  
Author(s):  
Synda Chenenaoui ◽  
Samia Daldoul ◽  
Ahmed Mliki
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Extraction Procedure ◽  
Rna Isolation ◽  
Extraction Methods ◽  
Purification Step ◽  
High Quality ◽  
Rna Quality ◽  
Expression Studies ◽  
Gene Expression Studies

AbstractObjectives:Grapevine root system plays a great role in sensing and adapting to abiotic and biotic stresses. Identification of candidate genes involved in the tolerance to abiotic stress is becoming a crucial strategy to select and breed resilient genotypes. However, obtaining high quality RNA from grapevine roots under hydroponic culture is difficult. Hence, we have developed a new extraction procedure to improve RNA quality for root gene expression studies.Methods:Conventional RNA extraction methods using CTAB are not suitable for gene expression studies and need to be improved. Here we report the application of a CTAB- based method for RNA extraction using an additional clean-up purification step.Results:The RIN value of the resulting RNA indicated that our procedure allowed the purification of high RNA quality and quantity. Hence, the clean-up purification step efficiently eliminated contaminants which inhibit downstream applications. Derived RNA was successfully used for differential gene expression analysis in salt stressed grapevine by Northern Blot hybridizations.Conclusion:In this study, we developed an efficient RNA isolation protocol from hydroponic cultivated grapevine roots which yielded RNA suitable for gene expression studies. This will open large perspectives in grapevine functional genomics with the identification of pertinent genes of agronomic interest.

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