scholarly journals An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens – sample preparation for epigenetic analysis

2019 ◽  
Author(s):  
Sebastian Rausch ◽  
Oliver Hasinger ◽  
Thomas König ◽  
Anne Schlegel ◽  
Gunter Weiss
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Analytical Techniques ◽  
High Volume ◽  
Magnetic Bead ◽  
Bisulfite Conversion ◽  
Manual Method ◽  
Automated Method ◽  
Biopsy Specimens

Abstract Objective: DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5mL of plasma or urine, using reagents from the commercially available Epi BisKit. Results: Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4x24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7h30min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized ß-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.

scholarly journals An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens – sample preparation for epigenetic analysis

2019 ◽  
Author(s):  
Sebastian Rausch ◽  
Oliver Hasinger ◽  
Thomas König ◽  
Anne Schlegel ◽  
Gunter Weiss
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Analytical Techniques ◽  
High Volume ◽  
Magnetic Bead ◽  
Bisulfite Conversion ◽  
Manual Method ◽  
Automated Method ◽  
Biopsy Specimens

Abstract Objective: DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5mL of plasma or urine, using reagents from the commercially available Epi BisKit. Results: Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4x24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7h30min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized ß-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.

scholarly journals An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens – sample preparation for epigenetic analysis

2019 ◽  
Author(s):  
Sebastian Rausch ◽  
Oliver Hasinger ◽  
Thomas König ◽  
Anne Schlegel ◽  
Gunter Weiss
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Analytical Techniques ◽  
High Volume ◽  
Magnetic Bead ◽  
Bisulfite Conversion ◽  
Manual Method ◽  
Automated Method ◽  
Biopsy Specimens

Abstract Objective: DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5mL of plasma or urine, using reagents from the commercially available Epi BisKit. Results: Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4x24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7h30min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized ß-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.

scholarly journals An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens – sample preparation for epigenetic analysis

2019 ◽  
Author(s):  
Sebastian Rausch ◽  
Oliver Hasinger ◽  
Thomas König ◽  
Anne Schlegel ◽  
Gunter Weiss
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Analytical Techniques ◽  
High Volume ◽  
Magnetic Bead ◽  
Bisulfite Conversion ◽  
Manual Method ◽  
Automated Method ◽  
Biopsy Specimens

Abstract Objective: DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5mL of plasma or urine, using reagents from the commercially available Epi BisKit. Results: Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4x24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7h30min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized ß-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.

scholarly journals An Automated and High-Throughput Immunoaffinity Magnetic Bead-Based Sample Clean-Up Platform for the Determination of Aflatoxins in Grains and Oils Using UPLC-FLD

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 583 ◽  
Author(s):  
Zhihong Xuan ◽  
Jin Ye ◽  
Bing Zhang ◽  
Li Li ◽  
Yu Wu ◽  
...  
Keyword(s):  
High Throughput ◽  
High Performance ◽  
Cost Effective ◽  
Magnetic Bead ◽  
The Novel ◽  
Analysis Method ◽  
Automated Method ◽  
Accuracy And Precision ◽  
Optimized Conditions

Sample clean-up remains the most time-consuming and error-prone step in the whole analytical procedure for aflatoxins (AFTs) analysis. Herein, an automated and high-throughput sample clean-up platform was developed with a disposable, cost-effective immunoaffinity magnetic bead-based kit. Under optimized conditions, the automated method takes less than 30 min to simultaneously purify 20 samples without requiring any centrifugation or filtering steps. When coupled to ultra-high performance liquid chromatography with fluorescence detection, this new analysis method displays excellent accuracy and precision as well as outstanding efficiency. Furthermore, an interlaboratory study was performed in six laboratories to validate the novel protocol. Mean recovery, repeatability, reproducibility, and Horwitz ratio values were within 91.9%–107.4%, 2.5%–7.4%, 2.7%–10.6%, and 0.26%–0.90, respectively. Results demonstrate that the developed sample clean-up platform is a reliable alternative to most widely adopted clean-up procedures for AFTs in cereals and oils.

scholarly journals DNA extraction and sequencing of the Mawangdui ancient cadaver protected by formalin

2020 ◽  
Author(s):  
Hua-Lin Huang ◽  
Shikui Yin ◽  
Huifang Zhao ◽  
Chao Tian ◽  
Jufang Huang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Magnetic Bead ◽  
Sequencing Data ◽  
Sequencing Library ◽  
Bead Method ◽  
Formalin Fixed ◽  
Better Than

AbstractMawangdui ancient Cadaver is the first wet corpse found in the world, which is famous for being immortal for over two thousands of years. After being unearthed, the female corpse was immersed in the formalin protective solution for more than 40 years. We used magnetic bead method and formalin fixed paraffing (FFPE) method to extract the DNA of the female corpse, respectively. PCR amplification, sanger sequencing, library building, high throughput sequencing (testing) and data processing were carried out on the DNA samples, and about 0.5% of the whole genome coverage sequencing data was obtained. Comparing the results of DNA trough two extraction and sequencing methods. We found that the FFPE and high throughput sequencing methods is better than others for DNA extraction of the ancient samples which were preserved in formalin, providing a guidance for dealing with formalin preserved ancient samples in the future.

scholarly journals Efficient Isolation of High-quality Total RNA from Strawberry

HortScience ◽  
2019 ◽  
Vol 54 (2) ◽  
pp. 380-384 ◽  
Author(s):  
Misaki Ishibashi ◽  
Takeshi Nabe ◽  
Yoko Nitta ◽  
Yuichi Uno
Keyword(s):  
High Throughput ◽  
Rna Isolation ◽  
Isoamyl Alcohol ◽  
Reproductive Organs ◽  
Throughput Capacity ◽  
High Quality ◽  
Rna Integrity ◽  
Manual Method ◽  
Automated Method ◽  
Nonionic Polymer

Sufficient yields of high-quality RNA are needed for next-generation sequencing and high-throughput real-time polymerase chain reaction analyses. In the case of strawberry (Fragaria ×ananassa) fruits, successful RNA isolation requires removal of abundant inhibitory substances (polysaccharides and polyphenols) that greatly reduce quality and yield. In this study, we applied various combinations of RNA isolation protocols directed at reproductive organs. The best manual isolation method involved nonionic polymer and modified acid guanidinium thiocyanate-phenol-chloroform treatments followed by phenol/chloroform/isoamyl alcohol extraction. Compared with other methods, this approach gave significantly higher yields [84.0 µg/g fresh weight (FW)] of RNA of greater purity (A260/A280 = 1.99; A260/230 = 1.51). Better-quality RNA (A260/230 = 2.11) was obtained using an automated method, but the yield was lower (18.1 µg/g FW) than that obtained manually. This automated method consisted of pretreatment with nonionic polymer followed by a silica-based system extraction. Although RNA of sufficient quality [RNA Integrity Number (RIN) ≥ 6.5 and 28S/18S ≥ 1.0] for RNA sequencing was obtained from receptacles using both automated and manual methods, the manual method yielded high-quality RNA from achenes and anthers. The automatic method features 6-fold faster high-throughput capacity, whereas the manual method has wider applicability to different tissues.

Elucidating the Weakly Reversible Cs-Pb-Br Perovskite Nanocrystal Reaction Network with High-Throughput Maps and Transformations

2020 ◽  
Author(s):  
Jakob Dahl ◽  
Xingzhi Wang ◽  
Xiao Huang ◽  
Emory Chan ◽  
Paul Alivisatos
Keyword(s):  
High Throughput ◽  
Dynamic Equilibrium ◽  
Reaction Network ◽  
Substantial Impact ◽  
Equilibrium Behavior ◽  
Automated Method ◽  
Lead Bromide ◽  
Different Shapes ◽  
Complex Chemistry ◽  
Lead Halide

<p>Advances in automation and data analytics can aid exploration of the complex chemistry of nanoparticles. Lead halide perovskite colloidal nanocrystals provide an interesting proving ground: there are reports of many different phases and transformations, which has made it hard to form a coherent conceptual framework for their controlled formation through traditional methods. In this work, we systematically explore the portion of Cs-Pb-Br synthesis space in which many optically distinguishable species are formed using high-throughput robotic synthesis to understand their formation reactions. We deploy an automated method that allows us to determine the relative amount of absorbance that can be attributed to each species in order to create maps of the synthetic space. These in turn facilitate improved understanding of the interplay between kinetic and thermodynamic factors that underlie which combination of species are likely to be prevalent under a given set of conditions. Based on these maps, we test potential transformation routes between perovskite nanocrystals of different shapes and phases. We find that shape is determined kinetically, but many reactions between different phases show equilibrium behavior. We demonstrate a dynamic equilibrium between complexes, monolayers and nanocrystals of lead bromide, with substantial impact on the reaction outcomes. This allows us to construct a chemical reaction network that qualitatively explains our results as well as previous reports and can serve as a guide for those seeking to prepare a particular composition and shape. </p>

Feasibility and Evaluation of Automated Methods for Radiolabeling of Radiopharmaceutical Kits with Gallium-68

2019 ◽  
Vol 12 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Alban Revy ◽  
François Hallouard ◽  
Sandrine Joyeux-Klamber ◽  
Andrea Skanjeti ◽  
Catherine Rioufol ◽  
...  
Keyword(s):  
The European Union ◽  
Preparation Time ◽  
Limit Of Quantification ◽  
Manual Method ◽  
Personnel Costs ◽  
Automated Method ◽  
Gallium 68 ◽  
The Right ◽  
First Time ◽  
Automated Methods

Objective: Recent gallium-68 labeled peptides are of increasing interest in PET imaging in nuclear medicine. Somakit TOC® is a radiopharmaceutical kit registered in the European Union for the preparation of [68Ga]Ga-DOTA-TOC used for the diagnosis of neuroendocrine tumors. Development of a labeling process using a synthesizer is particularly interesting for the quality and reproducibility of the final product although only manual processes are described in the Summary of Product (SmPC) of the registered product. The aim of the present study was therefore to evaluate the feasibility and value of using an automated synthesizer for the preparation of [68Ga]Ga-DOTA-TOC according to the SmPC of the Somakit TOC®. Methods: Three methods of preparation were compared; each followed the SmPC of the Somakit TOC®. Over time, overheads, and overexposure were evaluated for each method. Results: Mean±SD preparation time was 26.2±0.3 minutes for the manual method, 28±0.5 minutes for the semi-automated, and 40.3±0.2 minutes for the automated method. Overcost of the semi-automated method is 0.25€ per preparation for consumables and from 0.58€ to 0.92€ for personnel costs according to the operator (respectively, technician or pharmacist). For the automated method, overcost is 70€ for consumables and from 4.06€ to 6.44€ for personnel. For the manual method, extremity exposure was 0.425mSv for the right finger, and 0.350mSv for the left finger; for both the semi-automated and automated method extremity exposure were below the limit of quantification. Conclusion: The present study reports for the first time both the feasibility of using a [68Ga]- radiopharmaceutical kit with a synthesizer and the limits for the development of a fully automated process.

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