scholarly journals A high-throughput pipeline for scalable kit-free RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ping Han ◽  
Maybelle K. Go ◽  
Jeng Yeong Chow ◽  
Bo Xue ◽  
Yan Ping Lim ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Cost Effectiveness ◽  
High Throughput ◽  
Solid Phase ◽  
Rna Extraction ◽  
High Volume ◽  
Supply Chain Disruptions ◽  
Clinical Diagnoses ◽  
Volume Acquisition ◽  
Commercial Kit

AbstractAn overreliance on commercial, kit-based RNA extraction in the molecular diagnoses of infectious disease presents a challenge in the event of supply chain disruptions and can potentially hinder testing capacity in times of need. In this study, we adapted a well-established, robust TRIzol-based RNA extraction protocol into a high-throughput format through miniaturization and automation. The workflow was validated by RT-qPCR assay for SARS-CoV-2 detection to illustrate its scalability without interference to downstream diagnostic sensitivity and accuracy. This semi-automated, kit-free approach offers a versatile alternative to prevailing integrated solid-phase RNA extraction proprietary systems, with the added advantage of improved cost-effectiveness for high volume acquisition of quality RNA whether for use in clinical diagnoses or for diverse molecular applications.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rachelle Bester ◽  
Glynnis Cook ◽  
Johannes H. J. Breytenbach ◽  
Chanel Steyn ◽  
Rochelle De Bruyn ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Pathogen Detection ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Healthy Plant ◽  
Ion Torrent ◽  
Extraction Protocol ◽  
Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

scholarly journals Fast and Efficient 5′P Degradome Library Preparation for Analysis of Co-Translational Decay in Arabidopsis

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 466
Author(s):  
Marie-Christine Carpentier ◽  
Cécile Bousquet-Antonelli ◽  
Rémy Merret
Keyword(s):  
Gene Expression ◽  
Quality Control ◽  
Transcriptional Regulation ◽  
High Throughput ◽  
Library Preparation ◽  
Working Day ◽  
Set Up ◽  
Post Transcriptional Regulation ◽  
Commercial Kit

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5′monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5′P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5′P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control.

High-throughput solid-phase permethylation of glycans prior to mass spectrometry

2008 ◽  
Vol 22 (5) ◽  
pp. 721-734 ◽  
Author(s):  
Pilsoo Kang ◽  
Yehia Mechref ◽  
Milos V. Novotny
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Solid Phase

A high-throughput nanofibers mat-based micro-solid phase extraction for the determination of cationic dyes in wastewater

2016 ◽  
Vol 1460 ◽  
pp. 24-32 ◽  
Author(s):  
Feifei Qi ◽  
Liangliang Qian ◽  
Jingjing Liu ◽  
Xiaoqing Li ◽  
Lingeng Lu ◽  
...  
Keyword(s):  
Solid Phase Extraction ◽  
High Throughput ◽  
Solid Phase ◽  
Cationic Dyes ◽  
Phase Extraction

Analysis of Microbial Community Structure and Volatile Compounds in the Pit Mud Used for Manufacturing Taorong-type Baijiu Based on High-Throughput Sequencing

2021 ◽  
Author(s):  
Yanbo Liu ◽  
Mengxiao Sun ◽  
Pei Hou ◽  
Wenya Wang ◽  
Xiangkun Shen ◽  
...  
Keyword(s):  
Community Structure ◽  
Microbial Community ◽  
Qualitative Analysis ◽  
Volatile Compounds ◽  
High Throughput ◽  
Microbial Community Structure ◽  
High Throughput Sequencing ◽  
Solid Phase ◽  
Gas Chromatography Mass Spectrometry ◽  
Pit Mud

Abstract In this study, the pit mud used in manufacturing Taorong-type Baijiu was collected from the upper, middle, lower and bottom layers of pits in Henan Yangshao Liquor Co., LTD. Besides, high-throughput sequencing (HTS) technology was adopted to analyze the microbial community structure of the pit mud. In addition, the volatile compounds in the pit mud were subjected to preliminarily qualitative analysis through headspace-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The results of HTS demonstrated that there were 5, 3, 5 and 5 dominant bacterial phyla (including 11, 11, 9 and 8 dominant bacterial genera) and 3, 3, 3 and 3 dominant fungal phyla (including 4, 7, 7 and 5 dominant fungal genera) in the pit mud from F-S (upper), G-Z (middle), H-X (lower) and I-D (bottom), respectively. The qualitative analysis results of volatile compounds demonstrated that a total of 78 kinds of volatile compounds were detected in the pit mud, including 46, 45, 39 and 49 kinds in the pit mud from F-S, G-Z, H-X and I-D, respectively. Ester and acid were the two main components in the pit mud. Meanwhile, the correlation between microorganisms and main volatile compounds in the pit mud was analyzed. Moreover, Lentimicrobium, Syner-01 and Blvii28_wastewater-sludge group were found for the first time in the pit mud used for manufacturing Taorong-type Baijiu. The findings of this study could provide a theoretical foundation for improving the quality of pit mud and the flavor of Taorong-type Baijiu.

scholarly journals A High-Throughput Search for Composition–Phase–Property Relations of Cu–Ni/Ti–Al Elastic Copper Alloys

Materials ◽  
2018 ◽  
Vol 11 (12) ◽  
pp. 2513
Author(s):  
Wang Jianwei ◽  
Xiao Wei ◽  
Zhang Chuan ◽  
Sun Lu ◽  
Huang Guojie ◽  
...  
Keyword(s):  
High Throughput ◽  
Solid Phase ◽  
Copper Alloys ◽  
Ternary Systems ◽  
Diagram Method ◽  
Property Relations ◽  
Diffusion Layers ◽  
Heat Treated ◽  
Phase Property ◽  
Relationship Of

A high-throughput method was employed to effectively obtain the cross-scale relationship of elastic copper alloys. Firstly, a Cu–Ni–Ti–Cu25Al–Cu35Sn diffusion multiple was prepared and heat-treated under a specified condition to form a series of diffusion layers with the concentration gradient at the multielement metal interface. Then, the compositions, elastic moduli, and hardness of the Cu–Ni–Al and Cu–Ti–Al ternary copper alloys were tested. Meanwhile, the solid phase sequences in the diffusion zones were predicted by the CALPHAD (CALculation of PHAse Diagram) method. Through these experimental and calculated results, the composition–phase–property relations of the Cu–Ni–Al and Cu–Ti–Al ternary systems were established.

scholarly journals High-Throughput Analysis of Selected Urinary Hydroxy Polycyclic Aromatic Hydrocarbons by an Innovative Automated Solid-Phase Microextraction

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1869 ◽  
Author(s):  
Stefano Dugheri ◽  
Alessandro Bonari ◽  
Matteo Gentili ◽  
Giovanni Cappelli ◽  
Ilenia Pompilio ◽  
...  
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
High Throughput ◽  
Aromatic Hydrocarbons ◽  
High Throughput Screening ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
User Friendliness ◽  
High Throughput Analysis ◽  
Polycyclic Aromatic ◽  
User Friendly

High-throughput screening of samples is the strategy of choice to detect occupational exposure biomarkers, yet it requires a user-friendly apparatus that gives relatively prompt results while ensuring high degrees of selectivity, precision, accuracy and automation, particularly in the preparation process. Miniaturization has attracted much attention in analytical chemistry and has driven solvent and sample savings as easier automation, the latter thanks to the introduction on the market of the three axis autosampler. In light of the above, this contribution describes a novel user-friendly solid-phase microextraction (SPME) off- and on-line platform coupled with gas chromatography and triple quadrupole-mass spectrometry to determine urinary metabolites of polycyclic aromatic hydrocarbons 1- and 2-hydroxy-naphthalene, 9-hydroxy-phenanthrene, 1-hydroxy-pyrene, 3- and 9-hydroxy-benzoantracene, and 3-hydroxy-benzo[a]pyrene. In this new procedure, chromatography’s sensitivity is combined with the user-friendliness of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide on-fiber SPME derivatization using direct immersion sampling; moreover, specific isotope-labelled internal standards provide quantitative accuracy. The detection limits for the seven OH-PAHs ranged from 0.25 to 4.52 ng/L. Intra-(from 2.5 to 3.0%) and inter-session (from 2.4 to 3.9%) repeatability was also evaluated. This method serves to identify suitable risk-control strategies for occupational hygiene conservation programs.

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