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 An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260002
Author(s):  
María José Cárdenas Espinosa ◽  
Tabea Schmidgall ◽  
Georg Wagner ◽  
Uwe Kappelmeyer ◽  
Stephan Schreiber ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Bacterial Degradation ◽  
Rna Seq ◽  
High Quality ◽  
Total Rna ◽  
Aromatic Substrates ◽  
Next Generation Sequencing Ngs ◽  
Total Rna Extraction

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

scholarly journals A quick and cost-effective method for DNA-free total RNA isolation using magnetic silica beads

2020 ◽  
Author(s):  
Aniruddha Das ◽  
Debojyoti Das ◽  
Arundhati Das ◽  
Amaresh C. Panda
Keyword(s):  
Small Rna ◽  
Genomic Dna ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Quality ◽  
Total Rna ◽  
Rna Purification ◽  
Dna Contamination ◽  
Silica Beads ◽  
Magnetic Silica

ABSTRACTCurrent RNA purification methods widely use silica-based columns that allow quick isolation of high quality and right quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent for cell lysis, followed by precipitation of RNA using isopropanol resulted in a similar quantity and quality of RNA compared to the commercial TRIzol. The commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without the need for DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using a magnetic bead exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.

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 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.

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.

scholarly journals All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes

2019 ◽  
Vol 10 ◽  
Author(s):  
Ana Cláudia Silva ◽  
Virginia Ruiz-Ferrer ◽  
Ángela Martínez-Gómez ◽  
Marta Barcala ◽  
Carmen Fenoll ◽  
...  
Keyword(s):  
High Throughput ◽  
Genomic Dna ◽  
High Throughput Sequencing ◽  
Rna Extraction ◽  
High Quality ◽  
Total Rna ◽  
Total Rna Extraction

Microbial Diversity and Physicochemical Characteristics of the Maotai-Flavored Liquor Fermentation Process

2020 ◽  
Vol 20 (7) ◽  
pp. 4097-4109 ◽  
Author(s):  
Yijie Dai ◽  
Zhiqiang Tian ◽  
Wangni Meng ◽  
Zongjun Li
Keyword(s):  
Amylase Activity ◽  
Rna Extraction ◽  
Physicochemical Characteristics ◽  
Molecular Techniques ◽  
Its Sequencing ◽  
Total Rna ◽  
Microbial Community Structures ◽  
Rrna Sequencing ◽  
Fermented Grains ◽  
Dominant Bacteria

The grains fermented in Maotai-flavored liquor can be classified into two groups: high-temperature fermented grains generated by stacking and grains fermented in pits. The Maotai-flavor making process enriches a special microbiota from the fermented grains, which makes related studies more difficult. The use of modern molecular techniques to detect and analyze diversity and changes in total bacterial (16S rRNA sequencing) and fungal (internal transcribed spacer (ITS) sequencing) counts have helped to overcome the shortcomings of traditional technological research. The total RNA extracted by Fe3O4–SiO2 nanoparticles was retrieved into DNA by MagBeads Total RNA Extraction Kit. However, discrepancies exist in the microbial community structures at different fermentation periods. Dominant bacteria include Escherichia-Shigella, Lactobacillus, Clostridium, and Streptococcus species and dominant fungi include Alternaria, Ciliophora, Pyrenochaetopsis, Cyphellophora, Aspergillus, Issatchenkia, Pichia, Candida, and Zygosaccharomyces species. Analyses of the enzymatic activity of samples at different fermentation stages have revealed that some existing bacilli influence amylase activity. Here, to investigate flavor changes during each fermentation round, the liquor quality at all rounds was comprehensively assessed based on the corresponding physicochemical properties, which were summarized by sensory evaluation. These results suggested that the liquor quality in the third, fourth, and fifth fermentation rounds was superior.

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