Effective Isolation of High-Quality Total RNA from Human Adult Articular Cartilage

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.

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A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanense

PLoS ONE ◽

10.1371/journal.pone.0196592 ◽

2018 ◽

Vol 13 (5) ◽

pp. e0196592 ◽

Cited By ~ 12

Author(s):

Lulu Liu ◽

Rongchun Han ◽

Nianjun Yu ◽

Wei Zhang ◽

Lihua Xing ◽

...

Keyword(s):

High Quality ◽

Total Rna ◽

Dendrobium Huoshanense

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

Canadian Journal of Microbiology ◽

10.1139/w11-048 ◽

2011 ◽

Vol 57 (7) ◽

pp. 590-598 ◽

Cited By ~ 17

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.

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Improved protocol for isolation of high-quality total RNA from different organs of Phaseolus vulgaris L.

BioTechniques ◽

10.2144/btn-2018-0129 ◽

2019 ◽

Vol 66 (2) ◽

pp. 96-98 ◽

Cited By ~ 1

Author(s):

Alexis Acosta-Maspons ◽

Ingrid González-Lemes ◽

Alejandra A Covarrubias

Keyword(s):

Phaseolus Vulgaris ◽

Phaseolus Vulgaris L ◽

High Quality ◽

Total Rna

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Cartilage repair in the young knee

10.1093/med/9780199550647.003.008002 ◽

2011 ◽

Author(s):

Tim Spalding ◽

Lars Peterson

Keyword(s):

Articular Cartilage ◽

Cartilage Repair ◽

Clinical Analysis ◽

Short Term ◽

High Quality ◽

The Future ◽

Surgical Options

♦ Articular cartilage has a poor capacity to heal by itself♦ Left alone, large areas are likely to progress into osteoarthritis♦ The goal of cartilage repair is both short term improvement in function and long term durability♦ There are several available strategies including non-surgical options, and these are formulated into an algorithm♦ Cartilage repair is an advancing field and the future lies in bioengineering and high quality comparative clinical analysis.

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

Malaria Journal ◽

10.1186/s12936-020-03436-w ◽

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.

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