scholarly journals A Comprehensive RNA Handling and Transcriptomics Guide for High-Throughput Processing of Plasmodium Blood-Stage Samples.

2020 ◽  
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 ◽  
Infected Blood ◽  
Blood Samples ◽  
Total Rna

Abstract BackgroundSequencing 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, we present a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples that can be fully streamlined for high-throughput studies.Results We evaluated the utility of various commercially available RNA-preserving reagents in a range of storage conditions. Similarly, we compared several RNA extraction protocols and established the one most suitable for the extraction of high-quality total RNA from low-parasitemia and low-volume blood samples. Furthermore, we updated the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA. Optimization of SMART-seq2 amplification method to better suit AT-rich P. falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10ng of total RNA and a lower parasitemia limit of 0.05%. Finally, we designed a modified method for depletion of unwanted human hemoglobin transcripts using in vitro CRISPR-Cas9 treatment, thus improving parasite transcriptome coverage in low parasitemia samples. To prove the functionality of the pipeline for both laboratory and field strains, we generated the highest 2-hour resolution RNA-seq transcriptome for Plasmodium falciparum 3D7 intraerythrocytic lifecycle available up-to-date and also applied the entire protocol to create the largest transcriptome data from Southeast Asian field isolates.ConclusionsOverall, our methodology presents an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitemia 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 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 A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

2020 ◽  
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 BackgroundSequencing 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.ResultsThe 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 10ng 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 up-to-date was generated and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates.ConclusionsOverall, 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 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

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 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 High Throughput Phenotypic Screening of The Human Spermatozoon

Reproduction ◽  
2021 ◽  
Author(s):  
Zoe Claire Johnston ◽  
Franz S Gruber ◽  
Sean Brown ◽  
Neil R Norcross ◽  
Jason R Swedlow ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Hormonal Contraceptives ◽  
Diagnostic Tools ◽  
Sperm Function ◽  
Phenotypic Screening ◽  
Environmental Chemical ◽  
Potential Applications

Despite recent advances in male reproductive health research, there remain many elements of male (in)fertility where our understanding is incomplete. Consequently, diagnostic tools and treatments for men with sperm dysfunction, other than medically assisted reproduction, are limited. On the other hand, the gaps in our knowledge of the mechanisms which underpin sperm function have hampered the development of male non-hormonal contraceptives. The study of mature spermatozoa is inherently difficult. They are a unique and highly specialised cell type which does not actively transcribe or translate proteins and cannot be cultured for long periods of time or matured in vitro. One, large scale, approach to both increasing understanding of sperm function, and the discovery and development of compounds that can modulate sperm function, is to directly observe responses to compounds with phenotypic screening techniques. These target agnostic approaches can be developed into high-throughput screening platforms with the potential to drastically increase advances in the field. Here we discuss the rationale and development of high-throughput phenotypic screening platforms for mature human spermatozoa, and the multiple potential applications these present, as well as the current limitations and leaps in our understanding and capabilities needed to overcome them. Further development and use of these technologies could lead to the identification of compounds which positively or negatively affect sperm cell motility or function, or novel platforms for toxicology or environmental chemical testing among other applications. Ultimately, each of these potential applications is also likely to increase understanding within the field of sperm biology.

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.

High-Throughput Electrophysiology Screen Revealed Cardiotoxicity of Strychnine by Selectively Targeting hERG Channel

2018 ◽  
Vol 46 (08) ◽  
pp. 1825-1840 ◽  
Author(s):  
Taiyi Wang ◽  
Xiaonan Chen ◽  
Jiahui Yu ◽  
Qunqun Du ◽  
Jie Zhu ◽  
...  
Keyword(s):  
Herbal Medicine ◽  
Patch Clamp ◽  
High Throughput ◽  
Chinese Herbal Medicine ◽  
Large Scale ◽  
Cardiac Toxicity ◽  
Herg Channel ◽  
Chinese Herbal ◽  
Cell Vitality

Although the efficacy and the health care advantages of Chinese herbal medicine (CHM) have become increasingly recognized worldwide, the potential side effects and toxicity still restrict its broader application. This study established and applied an integrated platform anchored on automatic patch clamp system to screen and evaluate a collection of CHM extracts, compositions and monomeric compounds for in vitro cardiac toxicity. Of 1036 CHM samples screened, 2.79% significantly inhibited hERG channel activity. Among them, Strychnine was identified for the first time as a potent hERG inhibitor with an IC[Formula: see text] of [Formula: see text]M in comparison to that of Dofetilide at [Formula: see text]M and Quinidine at [Formula: see text]M. Langendorff-perfusion experiments confirmed that strychnine increased QT interphase from [Formula: see text][Formula: see text]ms to [Formula: see text][Formula: see text]ms and decreased heart rates from [Formula: see text][Formula: see text]bmp to [Formula: see text][Formula: see text]bmp in isolated rat hearts. The cardiac toxicity effect of strychnine appears to be specific to hERG channel since an in vitro multiplex imaging analysis showed that it did not affect cellular phenotypes such as cell vitality, nucleus area, mitochondria mass and function, nor intracellular calcium in rat primary myocytes. This integrated high-throughput hERG patch clamp and high-content multi-parameter imaging cardiac toxicity screen approach should be useful for large-scale preclinical evaluation of complex Chinese herbal medicine.

scholarly journals Feasibility of High-Throughput Genome-Wide Genotyping using DNA from Stored Buccal Cell Samples

2010 ◽  
Vol 5 ◽  
pp. BMI.S5062 ◽  
Author(s):  
Stephanie J. Loomis ◽  
Lana M. Olson ◽  
Louis R. Pasquale ◽  
Janey Wiggs ◽  
Daniel Mirel ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Buccal Cell ◽  
Health Study ◽  
Attractive Alternative ◽  
Sample Collection ◽  
Blood Samples ◽  
Dna Concentration ◽  
Genome Wide ◽  
Cell Sample

It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses' Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (>97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/μl. Most (>95%) genotype determinations in buccal cell samples were of the “missing call” variety (as opposed to the “alternative genotype call” variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform.

scholarly journals Molecular Fitness Landscapes from High-Coverage Sequence Profiling

2019 ◽  
Vol 48 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Celia Blanco ◽  
Evan Janzen ◽  
Abe Pressman ◽  
Ranajay Saha ◽  
Irene A. Chen
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Fitness Landscape ◽  
Complete Sequence ◽  
Fitness Landscapes ◽  
Future Research ◽  
High Coverage

The function of fitness (or molecular activity) in the space of all possible sequences is known as the fitness landscape. Evolution is a random walk on the fitness landscape, with a bias toward climbing hills. Mapping the topography of real fitness landscapes is fundamental to understanding evolution, but previous efforts were hampered by the difficulty of obtaining large, quantitative data sets. The accessibility of high-throughput sequencing (HTS) has transformed this study, enabling large-scale enumeration of fitness for many mutants and even complete sequence spaces in some cases. We review the progress of high-throughput studies in mapping molecular fitness landscapes, both in vitro and in vivo, as well as opportunities for future research. Such studies are rapidly growing in number. HTS is expected to have a profound effect on the understanding of real molecular fitness landscapes.

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