6,695 results match your criteria BioTechniques[Journal]
Biotechniques 2018 Dec;65(6):351-356
Human Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.
Antibody (Ab) repertoire sequencing using high-throughput massively parallel technologies has contributed substantially to the understanding of Ab responses following infection, vaccination and autoimmunity. Because individual B-cell receptors are recombined and diversified somatically, genomic comparisons are limited, and distinguishing rare variants from sequencing errors is a major challenge. Oxford Nanopore Technologies' MinION is a highly portable and cost-effective third-generation sequencing instrument, but has not been used for Ab repertoire sequencing due to its high error rate (approximately 1/10 bases). Read More
Biotechniques 2018 Dec;65(6):331-338
Division of Emerging & Transfusion-Transmitted Diseases, Laboratory of Bacterial & Transmissible Spongiform Encephalopathy Agents, Center for Biologics Evaluation & Research, Office of Blood Research & Review, US Food & Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA.
Staphylococcus epidermidis is the most common transfusion-associated pathogen contaminating platelet concentrates. Methods to reduce or eliminate contaminating bacteria from platelet units are critical for improving the safety of blood transfusions. We used rapid isolation of DNA aptamers (RIDA) to identify single-stranded (ss)DNA aptamers as ligands that specifically bind to S. Read More
Biotechniques 2018 Dec;65(6):357-360
Department of Epidemiology & Biostatistics, University of California, San Francisco, CA, USA.
Sample automation and management is increasingly important as the number and size of population-scale and high-throughput projects grow. This is particularly the case in large-scale population studies where sample size is far outpacing the commonly used 96-well plate format. To facilitate management and transfer of samples in this format, we present Samasy, a web-based application for the construction of a sample database, intuitive display of sample and batch information, and facilitation of automated sample transfer or subset. Read More
Biotechniques 2018 Dec;65(6):339-345
School of Biological Sciences, University of Northern Colorado, 501 20th Street, Greeley, CO 80639-0017, USA.
DNA barcoding is a simple technique used to develop a large-scale system of classification that is broadly applicable across a wide variety of taxa. DNA-based analysis of snake venoms can provide a system of classification independent of currently accepted taxonomic relationships by generating DNA barcodes specific to each venom sample. DNA purification from dried snake venoms has previously required large amounts of starting material, has resulted in low yields and inconsistent amplification, and was possible with front-fanged snakes only. Read More
Biotechniques 2018 Dec;65(6):303
Biotechniques 2018 Dec;65(6):322-330
Head & Neck Cancer Research Team, Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia.
We describe a novel automated cell detection and counting software, QuickCount (QC), designed for rapid quantification of cells. The Bland-Altman plot and intraclass correlation coefficient (ICC) analyses demonstrated strong agreement between cell counts from QC to manual counts (mean and SD: -3.3 ± 4. Read More
Biotechniques 2018 Dec;65(6):347-349
Department of Mechanical Engineering, Johns Hopkins University, MD, USA.
In this study, economic magnetic tweezers (EMT) with a sharp gradient field were designed and built, in order to facilitate accurate force measurement. Our design costs less than 40 USD and is easy to mount onto most microscope stages. We leverage the computational fluidic dynamics techniques to calculate the forces based on the results obtained using our simple device. Read More
Biotechniques 2018 Dec;65(6):312-314
Advanced Research Centre on Electronic Systems for Information & Communication Technologies 'E. De Castro' (ARCES), University of Bologna, Italy.
Biotechniques 2018 Nov;65(5):250-251
Department of Physics, Virginia Commonwealth University, Richmond, VA, USA.
Biotechniques 2018 Nov;65(5):253-257
Department of Biochemistry, Genetics & Microbiology, Tree Protection Co-operative Programme (TPCP), Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
It is challenging to sequence and assemble genomes of obligate plant pathogens and microorganisms because of limited amounts of DNA, comparatively large genomes and high numbers of repeat regions. We sequenced the 1.2 gigabase genome of an obligate rust fungus, Austropuccinia psidii, the cause of rust on Myrtaceae, with a Chromium 10X library. Read More
Biotechniques 2018 Nov;65(5):281-283
Department of Animal Science, University of California - Davis, Davis, CA, USA.
CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection. Read More
Biotechniques 2018 Nov;65(5):289-292
CultureTrax®, Cellara LLC, Madison, WI 53719, USA.
Cell culture is a vital component of laboratories throughout the scientific community, yet the absence of standardized protocols and documentation practice challenges laboratory efficiency and scientific reproducibility. We examined the effectiveness of a cloud-based software application, CultureTrax as a tool for standardizing and transferring a complex cell culture protocol. The software workflow and template were used to electronically format a cardiomyocyte differentiation protocol and share a digitally executable copy with a different lab user. Read More
Biotechniques 2018 Nov;65(5):285-287
Department of Molecular, Cellular & Biomedical Sciences, University of New Hampshire, 46 College Road, Durham, NH 03824, USA.
Whatman FTA Cards are a fast and efficient method for capturing and storing nucleic acids but can be cost-prohibitive for large numbers of samples. We developed a method that substitutes a readily-available cellulose matrix and homemade washing buffer for commercial FTA Cards and FTA Purification Reagent. This method is suitable for long-term storage of DNA from many plant species prior to PCR. Read More
Biotechniques 2018 Nov;65(5):275-280
Counterterrorism & Forensic Science Research Unit, Laboratory Division, Federal Bureau of Investigation, 2501 Investigation Parkway, Quantico, VA 22135, USA.
Described here is a novel, universal exogenous internal positive control (IPC), which is fully synthetic for unparalleled quality control. The IPC was rationally designed, is small and efficiently amplified, has been successfully utilized alone or in triplex qPCR reactions, and is not crossreactive to human DNA or to any of the numerous non-human DNA samples tested. It was sensitive to inhibition by at least four commonly encountered amplification inhibitors. Read More
Biotechniques 2018 Nov;65(5):243
Biotechniques 2018 Nov;65(5):269-274
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.
Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. Read More
Biotechniques 2018 Oct 29. Epub 2018 Oct 29.
Department of Obstetrics & Gynecology, Oregon Health & Science University, Portland, OR, USA.
Geometry of the placental villous vasculature is a key determinant of maternal-fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Read More
Biotechniques 2018 Oct 18. Epub 2018 Oct 18.
Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, Québec, H3G 1A4, Canada.
Single-cell analysis overcomes the problems of cellular heterogeneity by revealing the individual differences between cells in tissue. The current tools used to profile gene expression at the single-cell level are arduous and often require specialized equipment. We have previously developed a technique to quantify protein expression levels in single living cells. Read More
Biotechniques 2018 Oct;65(4):224-226
Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.
High-throughput protein expression platforms are increasingly used to produce proteins for many applications: to support studies in structure/function, regulation and proteomics, as well as for direct use as potential biotherapeutic agents for medical applications. Here we describe a device that we refer to as the flask density reader (FDR) consisting of a through-beam laser and sensor, and a customized culture flask-receiving nest. The FDR has been integrated onto GNF System™'s automated protein expression platform to enable rapid, noninvasive, fully automated spectrophotometric determination of cell densities in suspension mammalian cell cultures. Read More
Biotechniques 2018 Oct;65(4):197-203
Brain Korea 21 Plus Project for Biomedical Science, Korea University College of Medicine, Seoul 02841, Republic of Korea.
Isolation of spheroid-forming cells is important to investigate cancer stem cell (CSC) characteristics. However, conventional tumor spheroid culture methods have not proven suitable because the aggregated spheroids generally maintain their original heterogeneity and harbor multiple cells with various characteristics. Here we cultured spheroids using a polydimethylsiloxane microwell-based method and a limiting dilution protocol. Read More
Biotechniques 2018 Oct;65(4):211-218
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (IBT, UNAM), Avenida Universidad 2001, Col. Chamilpa, C.P. 62210, Cuernavaca, Morelos, México.
Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. Read More
Biotechniques 2018 Oct;65(4):187-189
Biomedical Institute for Global Health Research & Technology, National University of Singapore, Singapore 117599.
Biotechniques 2018 Oct;65(4):219-223
Department of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, PO Box 35, FI-40014, Finland.
Ribosomal RNA analysis is a useful tool for characterization of microbial communities. However, the lack of broad-range primers has hampered the simultaneous analysis of eukaryotic and prokaryotic members by amplicon sequencing. We present a complete workflow for directional, primer-independent sequencing of size-selected small subunit ribosomal RNA fragments. Read More
Biotechniques 2018 Oct;65(4):205-210
Canon US Life Sciences, Inc., 9800 Medical Center Drive Suite C-120, Rockville, MD 20850, USA.
We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol. Read More
Biotechniques 2018 Oct;65(4):179
Biotechniques 2018 Oct;65(4):191-196
Department of Stem Cell Biology & Regenerative Medicine, Broad Center for Regenerative Medicine & Stem Cell Research, Keck School of Medicine, University of Southern California, CA, USA.
The use of fluorescent tags to monitor protein expression and to lineage-trace cells has become a standard complement to standard histological techniques in the fields of embryology, pathology and regenerative medicine. Unfortunately, traditional paraffin embedding protocols can substantially diminish or abolish the native emission signal of the fluorophore of interest. To preserve the fluorescent signal, an alternative is to use cryosectioning; however, this can often result in undesirable artefacts such as tearing or shattering - particularly for mineralized tissues such as bone and cartilage. Read More
Biotechniques 2018 Oct;65(4):181-185
Alfie Gleeson and Abigail Sawyer explore the latest advances in high-throughput sequencing for neuroscience on the quest to map out the brain. High-throughput sequencing technology has enabled research centers across the globe to begin mapping neural connections and deciphering neural wiring circuits. From huge initiatives in the USA to smaller, but no less important research groups in Europe, the quest to map the brain is becoming a worldwide research goal. Read More
Biotechniques 2018 Sep;65(3):149-157
Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, LA 71201, USA.
Protein-protein interactions (PPI) by homo-, hetero- or oligo-merization in the cellular environment regulate cellular processes. PPI can be inhibited by antibodies, small molecules or peptides, and this inhibition has therapeutic value. A recently developed method, the proximity ligation assay (PLA), provides detection of PPI in the cellular environment. Read More
Biotechniques 2018 Sep;65(3):137-142
Faculty of Medicine, Institute of Medical, Pharmaceutical & Health Science, Kanazawa University, Takara-machi, Kanazawa 920-0934, Japan.
A modified invasion assay using a three-dimensional collagen gel was developed that enables isolation of invasive living cells; it was named the invading cell trapping (iCT) assay. A small cell strainer consisting of a nylon mesh with 40-μm pores was used, and collagen gel layers formed across the membrane. Test cells were seeded in the lower gel layer and invasive cells were attracted upward and trapped in the upper gel. Read More
Biotechniques 2018 Sep;65(3):111
Head of Open Access Publishing, Future Science Group, Unitec House, 2 Albert Place, London, UK.
Biotechniques 2018 Sep;65(3):125-126
Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Jena, 07745, Germany.
Biotechniques 2018 Sep;65(3):163-168
Institute of Genetics & Biotechnology, Faculty of Biology, University of Warsaw, A. Pawinskiego 5a, 02-106, Warsaw, Poland.
Comparative transcriptional analyses require appropriate and precise normalization. Here we describe a modified transcription run-on (TRO) method, named quantitative TRO (qTRO), that allows quantification of nascent transcription activity. The most critical improvement it introduces is a new standardization method for RNA isolation and hybridization steps, enabling transcription activity quantification and comparative biological analysis. Read More
Biotechniques 2018 Sep;65(3):159-162
Laboratoire de Microbiologie et de Génétique Moléculaires, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 31062 Toulouse Cedex 09, France.
A direct method to study essential genes is to construct conditional knock-down mutants by replacement of their native promoter by an inducible one. In Mycobacteria, replacement of an essential gene promoter with an anhydrotetracycline inducible one was successfully used but required a multi-step approach. In this work, we describe a gene cassette for the engineering of a conditional knock-down mutant, which allows the one-step targeted replacement of mycobacterial promoters by an anhydrotetracycline-inducible promoter. Read More
Biotechniques 2018 Sep;65(3):113-119
Cambridge University Synthetic Biology Society, University of Cambridge, CB2 3EA, UK.
[Formula: see text] Synthetic biology has enormous potential to solve problems in health, agriculture, and energy. Bill Jia and Arin Wongprommoon explore engineering approaches to controlling biological processes. Read More
Biotechniques 2018 Sep;65(3):143-148
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA.
Kinases are essential cell signaling enzymes that phosphorylate protein substrates using ATP as the universal cosubstrate. A wide variety of ATP analogs have been used in kinase research, although the studies are limited by the cell impermeability of ATP. Here we describe the use of the cationic polymer deacetylated chitosan to permeabilize ATP analogs for live cell applications, including kinase-catalyzed biotinylation. Read More
Biotechniques 2018 Dec 17;65(6):315-321. Epub 2018 Sep 17.
The Procter & Gamble Company, 8700 Mason Montgomery Road, Mason, OH 45040, USA.
Shotgun metagenomics is a powerful platform to characterize human microbiomes. However, to translate such survey data into consumer-relevant products or services, it is critical to have a robust metagenomics workflow. We present a tool - spike-in DNA - to assess performance of metagenomics workflows. Read More
Biotechniques 2018 Oct 24;65(4):227-230. Epub 2018 Aug 24.
Department of Microbiology, Kindai University Faculty of Medicine, Osakasayama, Osaka 589-8511, Japan.
Fecal occult blood (FOB) is a sign of gastrointestinal diseases, such as intestinal ulcers and colorectal cancer. In experimental animal studies, there is no standard method to detect FOB. Here, we present a simple protocol to detect FOB in mice, using the Luminol Reaction Experiment Kit that was originally designed to detect bloodstains at a crime scene in criminal forensics. Read More
Biotechniques 2018 Nov 17;65(5):259-267. Epub 2018 Aug 17.
Thermo Fisher Scientific Inc., R&D Synthetic Biology, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
GUIDE-seq was developed to detect CRISPR/Cas9 off-target. However, as originally reported, it was associated with a high level of nonspecific amplification. In an attempt to improve it, we developed target-enriched GUIDE-seq (TEG-seq). Read More
Biotechniques 2018 Aug;65(2):53
Biotechniques 2018 Aug;65(2):79-85
Servicio de Dermatología, Instituto Valenciano de Oncología, Valencia, Spain.
The use of NGS in clinical practice for precision diagnosis requires a quality starting material. Despite the broadly established use of formalin-fixed paraffin-embedded (FFPE) samples in molecular testing, these usually have low-quality DNA. We established a method to determine the suitability of melanoma FFPE samples for an amplicon-based NGS custom panel analysis. Read More
Biotechniques 2018 Aug;65(2):55-60
Biotechniques 2018 Aug;65(2):86-91
National Cancer Institute at the National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
Digital PCR has been promoted as a technique for obtaining absolute measures of the amount of nucleic acid target sequence in a sample, but still lacks standardization in data reporting. The initial method of representing data as copies per microliter produced inconsistent results and made inter-assay comparisons difficult. Normalizing copies to amount of nucleic acid gives more uniform results, but factors influencing the effective concentration of nucleic acid in the final digital PCR assay must be considered. Read More
Biotechniques 2018 Aug;65(2):97-100
Centre for Dermatology Research, School of Biological Sciences, Stopford Building, Oxford Road, University of Manchester, MAHSC & NIHR Biomedical Research Centre, Manchester, M13 9PT, UK.
RNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. Read More
Biotechniques 2018 Aug;65(2):93-95
MeMed, Tirat Carmel, Israel.
Distinguishing bacterial from viral infections is often challenging, leading to antibiotic misuse, and detrimental ramifications for the patient, the healthcare system and society. A novel ELISA-based assay that integrates the circulating levels of three host-response proteins (TRAIL, IP-10 and CRP) was developed to assist in differentiation between bacterial and viral etiologies. We developed a new protocol for measuring the host-based assay biomarkers using an automated ELISA workstation. Read More
Biotechniques 2018 Aug;65(2):101-104
Department of Retinal Vascular Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
In vivo imaging of mouse retinas using two-photon or confocal laser scanning fluorescence microscopy is a powerful tool for time-lapse analyses of the dynamic movements of cell populations. However, acute and reversible opacification of the crystalline lenses of mouse eyes under anesthesia decreases the visibility of the ocular fundus. Therefore, we developed a customized contact lens for preventing cataract during continuous retinal imaging in anesthetized mice. Read More
Biotechniques 2018 Aug;65(2):65-69
Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, Canada.