6,876 results match your criteria BioTechniques[Journal]


Discovery of alliin as a putative inhibitor of the main protease of SARS-CoV-2 by molecular docking.

Biotechniques 2020 May 27. Epub 2020 May 27.

College of Food Engineering, Jilin Agricultural Science and Technology University, Jilin, Jilin Province 132101, People's Republic of China.

The outbreak of viral pneumonia caused by the novel coronavirus SARS-CoV-2 that began in December 2019 caused high mortality. It has been suggested that the main protease (Mpro) of SARS-CoV-2 may be an important target to discover pharmaceutical compounds for the therapy of this life-threatening disease. Remdesivir, ritonavir and chloroquine have all been reported to play a role in suppressing SARS-CoV-2. Read More

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http://dx.doi.org/10.2144/btn-2020-0038DOI Listing

Genetic molecular markers to accelerate genetic gains in crops.

Biotechniques 2020 May 27. Epub 2020 May 27.

Center of Excellence in Genomics & Systems Biology, International Crops Research Institute for The Semi-Arid Tropics (ICRISAT), Hyderabad, India.

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http://dx.doi.org/10.2144/btn-2020-0066DOI Listing

The use of an inexpensive processing aid device (the Mouse PAD) to facilitate rodent tissue banking.

Biotechniques 2020 May 18. Epub 2020 May 18.

Division of Surgical Sciences, Department of Surgery, Duke University, Durham, NC 27710, USA.

The generation of an experimental animal model often requires considerable investment of both time and money. Typically, investigators are interested in specific organs and when experimental animals are euthanized, tissues that are not the focus of the research project are discarded. However, the remaining tissues from these animals could provide valuable scientific information if efficient, error-proof and economical approaches to collect and biobank them were available. Read More

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http://dx.doi.org/10.2144/btn-2019-0069DOI Listing

Circulating tumor cell clusters and circulating tumor cell-derived explant models as a tool for treatment response.

Biotechniques 2020 May 18. Epub 2020 May 18.

Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.

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http://dx.doi.org/10.2144/btn-2020-0029DOI Listing

, covers and coronavirus.

Authors:

Biotechniques 2020 May 18. Epub 2020 May 18.

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http://dx.doi.org/10.2144/btn-2020-0049DOI Listing

Profiling a human: the role of antibodies.

Authors:
Francesca Lake

Biotechniques 2020 May 18. Epub 2020 May 18.

Future Science Group, Unitec House, 2 Albert Pl, Finchley, London, N3 1QB, UK.

Francesca Lake explores the role of antibodies in the ongoing efforts looking to build an atlas of the human body and its diseases. Read More

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http://dx.doi.org/10.2144/btn-2020-0064DOI Listing

Optimization of staining with SYTO 9/propidium iodide: interplay, kinetics and impact on .

Biotechniques 2020 May 12. Epub 2020 May 12.

Guangdong Key Laboratory of Environmental Pollution & Health, School of Environment, Jinan University, Guangzhou, 510632, China.

Fluorophores SYTO 9 and propidium iodide (PI) are extensively applied in medicine, food industry and environmental monitoring to assess the viability of bacteria. However, the actual performance of these dyes remains largely unknown. In addition, their effects on the physiology of cells have not been elucidated. Read More

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http://dx.doi.org/10.2144/btn-2020-0036DOI Listing

Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA.

Biotechniques 2020 May 6. Epub 2020 May 6.

Epigenetics Research Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Read More

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http://dx.doi.org/10.2144/btn-2020-0008DOI Listing

Nanoadhesive layer to prevent protein absorption in a poly(dimethylsiloxane) microfluidic device.

Biotechniques 2020 May 6. Epub 2020 May 6.

Institute for Biochemistry, ETH Zürich, Zürich, CH 8093, Switzerland.

Poly(dimethylsiloxane) (PDMS) is widely used as a microfluidics platform material; however, it absorbs various molecules, perturbing specific chemical concentrations in microfluidic channels. We present a simple solution to prevent adsorption into a PDMS microfluidic device. We used a vapor-phase-deposited nanoadhesive layer to seal PDMS microfluidic channels. Read More

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http://dx.doi.org/10.2144/btn-2020-0025DOI Listing

A simple serum depletion method for proteomics analysis.

Biotechniques 2020 May 6. Epub 2020 May 6.

Clinical & Biomedical Proteomics Group, Leeds Institute of Medical Research, St James's University Hospital, Leeds, LS9 7TF, UK.

Serum is the body fluid most often used in biomarker discovery. Albumin, the most abundant serum protein, contributes approximately 50% of the serum protein content, with an additional dozen abundant proteins dominating the rest of the serum proteome. To profile this challenging protein mixture by proteomics, the abundant proteins must be depleted to allow for detection of the low-abundant proteins, the primary biomarker targets. Read More

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http://dx.doi.org/10.2144/btn-2020-0017DOI Listing

Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension.

Biotechniques 2020 May 6. Epub 2020 May 6.

Department of Molecular Biology & Genetics, Aarhus University, C.F. Møllers Allé 3, 8000 Aarhus C, Denmark.

We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair. Read More

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http://dx.doi.org/10.2144/btn-2019-0104DOI Listing

magnetic resonance imaging of orthotopic prostate cancer.

Biotechniques 2020 May 2. Epub 2020 May 2.

Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, 1400 Pressler St, Houston, TX 77030, USA.

Methods for imaging orthotopic prostate tumors within the prostate or small tumors with extension outside the prostate are needed to more closely model human prostate tumors, which are most commonly located within the gland or may extend just through the gland. By comparing MR sequences, we found that the T2-based Dixon 'water only' sequence best visualized tumors within the prostate of mouse models in both young and old mice and that tumor weight derived from this sequence correlated highly with tumor weight (r = 0.98, p < 0. Read More

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http://dx.doi.org/10.2144/btn-2020-0021DOI Listing

Emulsion PCR made easy.

Biotechniques 2020 Apr 27. Epub 2020 Apr 27.

Centre for Innovation in Infectious Disease Research, Education & Training (CIIDRET), University of Delhi South Campus, New Delhi, 110021, India.

Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. Read More

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http://dx.doi.org/10.2144/btn-2019-0161DOI Listing

Corrigendum.

Authors:

Biotechniques 2020 May 27;68(5):291. Epub 2020 Apr 27.

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http://dx.doi.org/10.2144/btn-2020-0999c1DOI Listing

Optimized methods for IL-17A refolding and anti-IL17A Fab production for co-crystallization with small molecules.

Biotechniques 2020 Apr 27. Epub 2020 Apr 27.

Discovery Project Unit, HitGen Inc., Chengdu, China.

Refolding of human interleukin 17A (IL-17A) has been reported; however, the key refolding protocol was not robust enough to deliver consistent results and to be easily scaled up for crystallization. Here we report an optimized refolding method for IL-17A. Although co-crystal structures of IL-17A with ligands have been obtained with a high-affinity peptide and an anti-IL-17A Fab as stabilizers, neither the production yield nor the characterization of the IL-17A/Fab complex was reported. Read More

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http://dx.doi.org/10.2144/btn-2019-0170DOI Listing

Real-time loop-mediated isothermal amplification assay for rapid detection of .

Biotechniques 2020 Apr 27. Epub 2020 Apr 27.

Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Via Madonna del Piano 10, 50019, Sesto Fiorentino (Firenze), Italy.

is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for using a field-deployable portable instrument. Read More

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http://dx.doi.org/10.2144/btn-2019-0168DOI Listing

AroCell TK 210 ELISA for determination of TK1 protein: age-related reference ranges and comparison with other TK1 assays.

Biotechniques 2020 Apr 27. Epub 2020 Apr 27.

AroCell AB, Virdings allé 32B, SE-754 50 Uppsala, Sweden.

Thymidine kinase 1 (TK1) is an enzyme involved in DNA precursor synthesis that has been used as a biomarker for prognosis and monitoring of different malignancies. In this study, we compared two immunoassays for measuring TK1 protein concentrations: the TK 210 ELISA (AroCell AB) and TK1 ELISA from Abcam. Overall, the TK 210 ELISA showed higher sensitivity than the Abcam TK1 ELISA for differentiating hematological malignancies (sensitivity of 0. Read More

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http://dx.doi.org/10.2144/btn-2019-0148DOI Listing

COVID-19: How has the scientific community risen to the challenge?

Authors:
Jenny Straiton

Biotechniques 2020 05 22;68(5):232-234. Epub 2020 Apr 22.

Future Science Group, Unitec House, 2 Albert Place, Finchley Central, London, N3 1QB, UK.

As the novel coronavirus SARS-CoV-2 spreads around the world, scientists have raced to develop vaccines and tests in order to curb the infection. Read More

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http://dx.doi.org/10.2144/btn-2020-0041DOI Listing

A method for isolating RNA from canine bone.

Biotechniques 2020 Apr 17. Epub 2020 Apr 17.

Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA.

Extracting sufficient quantity and quality RNA from bone is essential for downstream application, such as transcriptomic sequencing, to evaluate gene expression. Isolation of RNA from bone presents a unique challenge owing to the hypocellular, brittle and mineralized matrix, which makes homogenizing the tissue difficult and provides little RNA to work with. Removal of contaminating tissue, such as bone marrow and connective tissue, is essential for isolating RNA that is unique to osteoblasts, osteoclasts and osteocytes. Read More

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http://dx.doi.org/10.2144/btn-2019-0153DOI Listing

Cautionary note on the use of to study muscle phenotypes caused by mutations in the human gene.

Biotechniques 2020 Apr 17. Epub 2020 Apr 17.

Redox Homeostasis Group, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Sevilla, Spain.

Mutations in the human gene, encoding a slow skeletal muscle/β-cardiac myosin heavy chain, cause different types of myopathies. The nematode model has frequently been employed to study the molecular and physiological consequences of mutations in muscle function by introducing mutations into the gene, the worm ortholog. We report here that the model is not appropriate for such studies if they involve expression of the UNC-54 protein (wild-type or fused to green fluorescent protein) above endogenous levels. Read More

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http://dx.doi.org/10.2144/btn-2020-0012DOI Listing

Cost-effective storage solution for delivering umbilical cord with efficient isolation of mesenchymal stem cells.

Biotechniques 2020 Apr 14. Epub 2020 Apr 14.

Department of Immunology, Health Science Center, Shenzhen University, Shenzhen 518055, PR China.

Mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for treating many diseases. However, their proliferation and therapeutic abilities decline during the aging process and disease development. Therefore, fetal MSCs derived from the umbilical cord (UC) attract more attention. Read More

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http://dx.doi.org/10.2144/btn-2019-0146DOI Listing

A novel flow cytometry-based quantitative monocyte adhesion assay to estimate endothelial cell activation .

Biotechniques 2020 Apr 14. Epub 2020 Apr 14.

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

One of the earliest events in the development of atherosclerosis is endothelial activation, which is estimated at the functional level by quantifying monocyte adhesion. This involves the incubation of fluorescently labeled monocytes on top of cultured endothelial cells and quantifying the number of adhered monocytes. Currently, the quantification of adhered monocytes is done using microscopy or by lysing the cells and estimating the fluorescence. Read More

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http://dx.doi.org/10.2144/btn-2019-0169DOI Listing

Optimization of western blotting for the detection of proteins of different molecular weights.

Biotechniques 2020 Apr 14. Epub 2020 Apr 14.

Department of Sciences & Mathematics, Mississippi University for Women, Columbus, MS 39701, USA.

Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0-20%) in Towbin's transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. Read More

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http://dx.doi.org/10.2144/btn-2019-0124DOI Listing

TADBD: a sensitive and fast method for detection of typologically associated domain boundaries.

Biotechniques 2020 Apr 7. Epub 2020 Apr 7.

School of Electronic & Information Engineering, Xi'an Jiaotong University, Xi'an 710049, China.

A topologically associated domain (TAD) is a self-interacting genomic block. Detection of TAD boundaries on Hi-C contact matrix is one of the most important issues in the analysis of 3D genome architecture at TAD level. Here, we present TAD boundary detection (TADBD), a sensitive and fast computational method for detection of TAD boundaries on Hi-C contact matrix. Read More

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http://dx.doi.org/10.2144/btn-2019-0165DOI Listing

A novel method for removing polyethyleneimine from biopharmaceutical samples: improving assay sensitivity of residual DNA qPCR.

Biotechniques 2020 Mar 31. Epub 2020 Mar 31.

Structure & Function Characterization, CMC Analytical, GlaxoSmithKline, Collegeville, PA, USA.

Polyethyleneimine (PEI) is a flocculent that is widely used in the downstream purification of monoclonal antibodies. It is an in-process residual that is carried through the drug purification process and strongly inhibits residual DNA quantitation by real-time quantitative PCR assay. Very high sample dilutions (e. Read More

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http://dx.doi.org/10.2144/btn-2020-0011DOI Listing

Examination of vesicular stomatitis virus-induced morphology changes in individual Vero cells by QMod microscopy.

Biotechniques 2020 Mar 23. Epub 2020 Mar 23.

Process Development, Ology Bioservices, Alachua, FL 32615, USA.

Viral infection of cultured cells induces changes in the biophysical characteristics of the affected cells. Advanced microscopic cameras such as Ovizio's QMod, coupled with the appropriate software, can measure a variety of characteristics on a per-cell basis. We have employed this system to monitor the progression of vesicular stomatitis virus infection in Vero cells and to describe the cellular changes associated with advancing vesicular stomatitis virus infection. Read More

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http://dx.doi.org/10.2144/btn-2019-0137DOI Listing

Big data, big future.

Authors:
Joseph Martin

Biotechniques 2020 Apr 23;68(4):166-168. Epub 2020 Mar 23.

Future Science Group, Unitec House, 2 Albert Place, Finchley Central, N3 1QB, London, UK.

With our increasing capabilities in research techniques and tools comes an inevitable increase in the amount of data and the complexity of datasets. But how do we cope with this plethora of data? Read More

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http://dx.doi.org/10.2144/btn-2020-0027DOI Listing

Improving RNAi efficiency for pest control in crop species.

Biotechniques 2020 May 23;68(5):283-290. Epub 2020 Mar 23.

Department of Entomology, MOA Key Lab of Pest Monitoring & Green Management, College of Plant Protection, China Agricultural University, Beijing, People's Republic China.

The application of RNAi promotes the development of novel approaches toward plant protection in a sustainable way. Genetically modified crops expressing dsRNA have been developed as commercial products with great potential in insect pest management. Alternatively, some nontransformative approaches, including foliar spray, irrigation and trunk injection, are favorable in actual utilization. Read More

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http://dx.doi.org/10.2144/btn-2019-0171DOI Listing

Cellular considerations for optimizing bone cell culture and remodeling in a lab-on-a-chip platform.

Biotechniques 2020 May 9;68(5):263-269. Epub 2020 Mar 9.

Department of Biomedical Engineering, The University of Akron, Akron, OH 44325, USA.

Our lab has developed a lab-on-a-chip platform for bone remodeling that enables long-term culturing of bone cells out to 7 weeks and serves as a foundation toward a multicellular organ-on-a-chip system. Here, we optimized culturing protocols for osteoblasts, osteoclasts and osteocytes within the lab-on-a-chip and performed functional activity assays for quantifying bone formation and resorption. We analyzed cell seeding densities, feeding schedules and time in culture as a basis for optimizing culturing protocols. Read More

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http://dx.doi.org/10.2144/btn-2019-0115DOI Listing

Intratracheal aerosolization of viral vectors to newborn pig airways.

Biotechniques 2020 May 6;68(5):235-239. Epub 2020 Mar 6.

Stead Family Department of Pediatrics, Pappajohn Biomedical Institute, & Center for Gene Therapy, The University of Iowa, Iowa City, IA 52242, USA.

Gene therapy for airway diseases requires efficient delivery of nucleic acids to the airways. In small animal models, gene delivery reagents are commonly delivered as a bolus dose. However, large animal models are often more relevant for the transition from preclinical studies to human trials. Read More

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http://dx.doi.org/10.2144/btn-2019-0150DOI Listing

Influence of spore morphology on spectrophotometric quantification of inocula.

Biotechniques 2020 May 6;68(5):279-282. Epub 2020 Mar 6.

Department of Food Sciences & Technology, BOKU-University of Natural Resources & Life Sciences, Vienna, Austria.

Species of the genus are filamentous fungi commonly used in research, industry and agriculture. strains are prominent producers of cellulolytic and hemicellulolytic enzymes as well as being expression hosts; several other species such as might be exploited as biocontrol agents. A careful preparation of inocula, which consists mainly of conidia (asexual spores), is of immense importance. Read More

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http://dx.doi.org/10.2144/btn-2019-0152DOI Listing

Development of an automated AmpliSeq™ library building workflow for biological stain samples on the Biomek 3000.

Biotechniques 2020 Mar 6. Epub 2020 Mar 6.

Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health & Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Here, we present the development of an automated AmpliSeq™ (ThermoFischer, MA, USA) workflow for library building using the Biomek 3000 Laboratory Automation Workstation (Beckman Coulter Inc., CA, USA), in which the total volume of PCR reagents and reagents for library preparation are reduced by one-half. The automated AmpliSeq workflow was tested using 43 stain samples (blood, bone, muscle tissue, semen, swab, nail scrape and cigarette butts) collected from crime scenes. Read More

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http://dx.doi.org/10.2144/btn-2019-0156DOI Listing

A method combining TA cloning and fluorescence screening for rapid acquisition of transgenic seeds.

Biotechniques 2020 May 6;68(5):251-256. Epub 2020 Mar 6.

Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Tropical Crops, Hainan University, Haikou 570228, China.

The establishment of transgenic plants has greatly promoted the progress of plant research. However, traditional selection methods using antibiotics or herbicides may miss any positive transformants with growth defects. Additionally, screening with antibiotics/herbicides requires a huge amount of seeds, sterile work conditions and a large amount of space to germinate plants, making the selection process time- and labor-consuming. Read More

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http://dx.doi.org/10.2144/btn-2019-0141DOI Listing
May 2020
2.948 Impact Factor

Improved third-generation lentiviral packaging with pLKO.1C vectors.

Biotechniques 2020 Mar 6. Epub 2020 Mar 6.

The Vision Center, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.

A widely used third-generation lentiviral packaging system produces virus with enhanced biosafety by eliminating HIV accessory genes and separating packaging elements into three different plasmids. However, for certain vectors such as pLKO.1, third-generation safety features reduce lentiviral titers due to the lack of the accessory gene . Read More

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http://dx.doi.org/10.2144/btn-2019-0155DOI Listing

A dextran-based warming method for preparing leukocyte-rich plasma and its clinical application for endotoxin assay.

Biotechniques 2020 Mar 2. Epub 2020 Mar 2.

Department of Critical Care Medicine, Iwate Medical University, Morioka, Japan.

We devised a method using dextran for obtaining leukocyte-rich plasma (LRP) to measure endotoxin in blood. In order to find the optimal temperature for obtaining LRP, the measurement results were examined using samples prepared at 37 and 0°C. Sample separation time of LRP was significantly shorter at 37°C than at 0°C. Read More

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http://dx.doi.org/10.2144/btn-2020-0005DOI Listing

Nanoliter-scale next-generation sequencing library-mediated high-throughput 16S rRNA microbial community profiling.

Biotechniques 2020 Apr 25;68(4):204-210. Epub 2020 Feb 25.

Anhui MicroAnaly Gene Technologies Co., Ltd, Chaohu, Anhui, China.

An ultra-high-throughput workflow for next-generation sequencing library construction at nanoliter scale for amplicon sequencing, termed Smartchip Nanowell Platform for Target Enrichment, was established using a nanodispenser system and a nanoliter-scale PCR chip. To demonstrate its cost and time advantages over conventional methods for library construction, quality control and pooling for large-scale samples, target amplicon sequencing of the 16S ribosomal RNA gene V3-V4 region widely used for microbial community profiling was chosen for comparison. The finding of no significant difference in microbial community profiling between the two methods strongly supports the conclusion that Smartchip Nanowell Platform for Target Enrichment is a cost-effective method for next-generation sequencing library construction for large-scale samples to conduct amplicon sequencing-based applications. Read More

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http://dx.doi.org/10.2144/btn-2019-0102DOI Listing
April 2020
2.948 Impact Factor

3D-printable cell crowding device enables imaging of live cells in compression.

Biotechniques 2020 May 25;68(5):275-278. Epub 2020 Feb 25.

Department of Mechanical Engineering, University of California, Santa Barbara, CA 93106, USA.

We designed and fabricated, using low-cost 3D printing technologies, a device that enables direct control of cell density in epithelial monolayers. The device operates by varying the tension of a silicone substrate upon which the cells are adhered. Multiple devices can be manufactured easily and placed in any standard incubator. Read More

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http://dx.doi.org/10.2144/btn-2019-0160DOI Listing

A cell-based assay for the detection of neutralizing antibodies against alemtuzumab.

Biotechniques 2020 Apr 25;68(4):185-190. Epub 2020 Feb 25.

Centre for Oral Immunobiology & Regenerative Medicine, Institute of Dentistry, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, London, E1 2AT, UK.

The humanized anti-CD52 monoclonal antibody alemtuzumab depletes lymphocytes and is currently used to treat relapsing multiple sclerosis. During treatment, anti-alemtuzumab antibodies may develop and reduce effective lymphocyte depletion in future treatment cycles. Alemtuzumab-Alexa Fluor 488 conjugate binding to the CHO-CD52 cell surface was inhibited by anti-alemtuzumab antibodies. Read More

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http://dx.doi.org/10.2144/btn-2019-0122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177201PMC

Isolation of extracellular vesicles from intestinal tissue in a mouse model of intestinal ischemia/reperfusion injury.

Biotechniques 2020 May 24;68(5):257-262. Epub 2020 Feb 24.

Department of Anesthesiology, Nanfang Hospital, Southern Medical University, Guangzhou, China.

Extracellular vesicles (EVs) are small membranous particles that contribute to intercellular communications. Separating EVs from tissue is still a technical challenge. Here, we present a rigorous method for extracting EVs from intestinal tissue in a mouse intestinal ischemia/reperfusion (I/R) model, and for analyzing their miRNA content. Read More

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http://dx.doi.org/10.2144/btn-2019-0159DOI Listing

Phagocytosis assays with different pH-sensitive fluorescent particles and various readouts.

Biotechniques 2020 May 21;68(5):245-250. Epub 2020 Feb 21.

Drug Discovery Science, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss 88397, Germany.

Phagocytosis is a fundamental mechanism of innate immunity and its impairment is associated with severe chronic diseases, for example, chronic obstructive pulmonary disease. Investigating phagocytosis requires flexible tools and assay conditions, such as different fluorescent particle types, detection colors and readouts. We comprehensively evaluated and optimized phagocytosis assays using particles labeled with fluorescent pH-sensitive pHrodo dyes, facilitating the specific detection of phagocytosed particles. Read More

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http://dx.doi.org/10.2144/btn-2020-0003DOI Listing

A systematic investigation of key factors of nucleic acid precipitation toward optimized DNA/RNA isolation.

Biotechniques 2020 Apr 18;68(4):191-199. Epub 2020 Feb 18.

Henan Key Laboratory of Ion-beam Bioengineering, School of Physics, Zhengzhou University, Zhengzhou 450000, China.

Nucleic acid precipitation is important for virtually all molecular biology investigations. However, despite its crucial role, a systematic study of the influence factors of nucleic acid precipitation has not been reported. In the present work, via rational experimental design, key factors of nucleic acid precipitation, including the type of nucleic acid, temperature and time of incubation, speed and time of centrifugation, volume ratio of ethanol/isopropanol to nucleic acid solution, type of cation-containing salt solution and type of coprecipitator, were comprehensively evaluated in an attempt to maximize the efficiency of nucleic acid precipitation. Read More

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http://dx.doi.org/10.2144/btn-2019-0109DOI Listing
April 2020
2.948 Impact Factor

Conventional and innovative methods to assess oxidative stress biomarkers in the clinical cardiovascular setting.

Biotechniques 2020 Apr 18;68(4):223-231. Epub 2020 Feb 18.

Fondazione CNR-Regione Toscana G Monasterio, Pisa 56100, Italy.

Oxidative stress has a pivotal and widely described role in the onset and progression of atherosclerotic plaque and cardiovascular disease. Many oxidative stress-related biomarkers can be measured in biological samples; however, there are still many aspects that limit the adoption of oxidative stress assessment in clinical laboratory practice. Here, we report an overview of the different sources and main common oxidative stress biomarkers relevant for cardiovascular physiopathology, describing in detail a recently proposed lipidomic assay for ceramide assessment, as a promising future development in oxidative stress evaluation among the many available redox-related tests. Read More

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http://dx.doi.org/10.2144/btn-2019-0138DOI Listing
April 2020
2.948 Impact Factor

A simple and reliable method for determination of optimum pH in coupled enzyme assays.

Biotechniques 2020 Apr 14;68(4):200-203. Epub 2020 Feb 14.

Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Here, we demonstrate a simple and reliable method to determine the pH optimum for pyruvate kinase using the pyruvate kinase/lactate dehydrogenase coupled enzyme assay. This simple and reliable method can be broadly adapted to determine the pH optimum for various enzymes that are assayed using a coupled enzyme assay. Read More

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http://dx.doi.org/10.2144/btn-2019-0126DOI Listing

A rapid and simple bead-bashing-based method for genomic DNA extraction from mammalian tissue.

Biotechniques 2020 May 14;68(5):240-244. Epub 2020 Feb 14.

Department of Obstetrics & Gynecology, Columbia University Irving Medical Center, New York, NY 10032, USA.

Conventional genomic DNA (gDNA) extraction methods can take hours to complete, may require fume hoods and represent the most time-consuming step in many gDNA-based molecular assays. We systematically optimized a bead bashing-based (BBB) approach for rapid gDNA extraction without the need for a fume hood. Human tissue specimens (n = 34) subjected to the 12-min BBB method yielded 0. Read More

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http://dx.doi.org/10.2144/btn-2019-0172DOI Listing

Developing new ways to diagnose cancer.

Biotechniques 2020 03 14;68(3):111-113. Epub 2020 Feb 14.

Future Science Group, Unitec House, 2 Albert Place, N3 1QB, London, UK.

We explore the latest developments across cancer diagnostic techniques that are enabling researchers to improve accuracy in determining diagnosis and prognosis. Read More

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http://dx.doi.org/10.2144/btn-2020-0014DOI Listing

Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species .

Biotechniques 2020 Apr 10;68(4):172-179. Epub 2020 Feb 10.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA 92697-4025, USA.

CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito : Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Read More

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http://dx.doi.org/10.2144/btn-2019-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177198PMC

Cellular thermal shift analysis for interrogation of CRISPR-assisted proteomic changes.

Biotechniques 2020 Apr 10;68(4):180-184. Epub 2020 Feb 10.

John Wayne Cancer Institute & Pacific Neuroscience Institute, Providence Saint John's Health Center, Santa Monica, CA 90404, USA.

CRISPR-Cas9 has proven to be a versatile tool for the discovery of essential genetic elements involved in various disease states. CRISPR-assisted dense mutagenesis focused on therapeutically challenging protein complexes allows us to systematically perturb protein-coding sequences and correlate them with functional readouts. Such perturbations can mimic targeting by therapeutics and serve as a foundation for the discovery of highly specific modulators. Read More

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http://dx.doi.org/10.2144/btn-2019-0100DOI Listing
April 2020
2.948 Impact Factor

Noninvasive prenatal diagnosis of hemophilia A by a haplotype-based approach using cell-free fetal DNA.

Biotechniques 2020 03 30;68(3):117-121. Epub 2020 Jan 30.

BGI Genomics, BGI-Shenzhen, Bei Shan Industrial Zone, Yantian District, Shenzhen 518083, Guangdong, China.

We aimed to demonstrate noninvasive prenatal diagnosis (NIPD) of hemophilia A (HA) using a haplotype-based approach. Two families at risk for HA were recruited for this study. First, maternal haplotypes associated with pathogenic variants were constructed using the genotypes of the mothers and probands. Read More

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http://dx.doi.org/10.2144/btn-2019-0113DOI Listing

Evaluation of DNA degradation and establishment of a degradation analysis model for Lepidoptera specimens.

Biotechniques 2020 03 28;68(3):138-147. Epub 2020 Jan 28.

Key Laboratory of Forest Protection of China State Forestry Administration, Research Institute of Forest Ecology, Environment & Protection, Chinese Academy of Forestry, No. 1 Dongxiaofu, Haidian District, Beijing 100091, China.

Millions of museum specimens are integral to biodiversity studies; however, DNA degradation may limit the ability to obtain DNA sequences. In this study, a degradation analysis model for Lepidoptera specimens was established. Based on this model, we revealed the characteristics of DNA fragment distribution caused by external DNA damage factors during specimen preservation. Read More

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http://dx.doi.org/10.2144/btn-2019-0166DOI Listing

Genomic DNA PCR analysis to assess xenograft development in mouse mammary gland.

Biotechniques 2020 Apr 28;68(4):219-222. Epub 2020 Jan 28.

Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350, Jouy-en-Josas, France.

The mouse transplantation model remains the most relevant methodology to assess the functional capacities of mammary cells and is particularly appropriate for investigations regarding mammary stem cells, whatever the species studied. Following xenotransplantation in mice mammary fat pad, the development of the xenograft is commonly evaluated by immunohistology. Here, we present a simple and rapid method to control the species specificity of a xenograft based on genomic DNA PCR amplification. Read More

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http://dx.doi.org/10.2144/btn-2019-0125DOI Listing