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    1 OF 132

    MicroScale Thermophoresis (MST) for studying actin polymerization kinetics.
    Biotechniques 2017 Oct 1;63(4):187-190. Epub 2017 Oct 1.
    Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
    Here, we present a MicroScale Thermophoresis (MST)-based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation. Read More

    Genotyping live fish larvae: Non-lethal and noninvasive DNA isolation from 3-5 day old hatchlings.
    Biotechniques 2017 Oct 1;63(4):181-186. Epub 2017 Oct 1.
    Department of Marine Biology, Texas A&M University, Galveston Campus, Galveston, TX; Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX.
    Genotyping fish larvae is a valuable technique for numerous fields of study. While methods for collecting DNA from early stage larvae have been published, a non-lethal, non-invasive genotyping protocol for hatchlings that is amenable to high-throughput approaches is desirable. Here, we describe a method to individually genotype live, free-swimming, early fish larvae by characterizing their environmental DNA (eDNA). Read More

    An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.
    Biotechniques 2017 Oct 1;63(4):174-180. Epub 2017 Oct 1.
    Dokuz Eylul University, School of Medicine, Department of Medical Biochemistry, Izmir, Turkey.
    Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Read More

    Density separation of quiescent yeast using iodixanol.
    Biotechniques 2017 Oct 1;63(4):169-173. Epub 2017 Oct 1.
    Department of Biology.
    As yeast are starved of nutrients, they enter G0, a quiescent state. Quiescent yeast (Q) cells retain viability for extended periods of time and resume growth following supplementation of missing nutrients. As such, Q cells have become a valuable model for studying longevity and self-renewal of chronologically aged cells. Read More

    Image-based cell-size estimation for baculovirus quantification.
    Biotechniques 2017 Oct 1;63(4):161-168. Epub 2017 Oct 1.
    University of Tartu, Institute of Chemistry, Tartu, Estonia.
    Measurement of virus concentration is essential for effective virus-based transfection technologies. Here, we describe a user-friendly, image-based cell-size estimation (ICSE) assay for baculovirus quantification that relies on automated determination of cell diameters from bright-field microscopy images. In the ICSE assay, microplate-based imaging systems and our custom ICSE-Tools software enable measurement of cell morphological parameters over time. Read More

    Development and Optimization of a High Titer Recombinant Lentivirus System.
    Biotechniques 2017 Sep 1;63(3):136-138. Epub 2017 Sep 1.
    Mirus Bio LLC, Madison, Wisconsin, USA.
    To enable simple and effective high titer recombinant lentivirus production, we examined key parameters for the generation of lentivirus including: transfection optimization, media change, incubation time and DNA vector selection. These results illustrate the importance of optimizing transfection processes for high titer recombinant lentivirus production. Read More

    Generation of iPS-derived model cells for analyses of hair shaft differentiation.
    Biotechniques 2017 Sep 1;63(3):131-134. Epub 2017 Sep 1.
    Department of Biomedical Chemistry, Graduate School of Science and Technology, Kwansei Gakuin University, 669-1337, Sanda, Japan.
    Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). Read More

    Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning.
    Biotechniques 2017 Sep 1;63(3):125-130. Epub 2017 Sep 1.
    Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea.
    Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. Read More

    Direct quantification of protein glycan phosphorylation.
    Biotechniques 2017 Sep 1;63(3):117-123. Epub 2017 Sep 1.
    Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research (CDER), Food and Drug Administration (FDA), Silver Spring, MD.
    Phosphorylation is an important post-translational modification (PTM) of proteins and a critical quality attribute for protein therapeutics, especially if it is required for protein function or sub-cellular targeting. Most current methods to quantify phosphorylation are time-consuming, indirect, or require specific instrumentation and technical skills. Here, we report the adaptation of a phosphate-specific binding dye and common laboratory instruments for quantification of relative amounts of phosphorylated glycans as well as phosphorylation of amino acid residues on the backbones of proteins. Read More

    Construction of a combinatorial library of chimeric tumor-specific promoters.
    Biotechniques 2017 Sep 1;63(3):107-116. Epub 2017 Sep 1.
    Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
    Gene therapy is a fast-developing field of molecular medicine. New, effective, and cancer-specific promoters are in high demand by researchers seeking to treat cancer through expression of therapeutic genes. Here, we created a combinatorial library of tumor-specific chimeric promoter modules for identifying new promoters with desired functions. Read More

    [Letter to the Editor] Incorrect assignment of affected nucleotides in footprinting/probing experiments.
    Biotechniques 2017 Sep 1;63(3):105-106. Epub 2017 Sep 1.
    Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
    Address correspondence to Sergey Belikov or Lars Wieslander, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: sergey.belikov@su. Read More

    Overcoming qRT-PCR interference by select carbon nanotubes in assessments of gene expression.
    Biotechniques 2017 Aug 1;63(2):81-84. Epub 2017 Aug 1.
    Department of Environmental and Global Health, University of Florida.
    Nanomaterials (NMs) of various types, including carbon nanotubes (CNTs), can interfere with standard quantitative real-time PCR (qRT-PCR) assays, resulting in inaccurate gene expression measurements; however, the precise step in the qRT-PCR pipeline where this interference occurs has not been well described. Here, we investigated where in the process surface-oxidized multi-walled CNTs (oxMWNTs) inhibited qRT-PCR measurement of the expression of the housekeeping gene GAPDH and explored several strategies to minimize such inhibition. We determined that the interference occurred during the reverse transcription (RT) step and found that doubling reaction reagents or adding BSA successfully mitigated the inhibition. Read More

    BODIPY-based dye for no-wash live-cell staining and imaging.
    Biotechniques 2017 Aug 1;63(2):77-80. Epub 2017 Aug 1.
    M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russian Federation.
    In nonpolar solvents, hydrophobic organic fluorophores often show bright fluorescence, whereas in polar media, they usually suffer from aggregation-caused quenching (ACQ). Here, we harnessed this solvatochromic behavior of a 1,3,5,7-tetramethyl-BODIPY derivative for cell staining and applied it to live-cell imaging and flow cytometry. As opposed to commercially available dyes, this BODIPY derivative showed excellent contrast immediately after staining and did not require any wash-off. Read More

    Spin infection enables efficient gene delivery to muscle stem cells.
    Biotechniques 2017 Aug 1;63(2):72-76. Epub 2017 Aug 1.
    Stem Cell Institute.
    Viral vector-mediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. Read More

    Ion-exchange chromatography purification of extracellular vesicles.
    Biotechniques 2017 Aug 1;63(2):65-71. Epub 2017 Aug 1.
    Institute for the Application of Nuclear Energy, INEP, University of Belgrade, Belgrade, Serbia.
    Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. Read More

    Preparation of highly multiplexed small RNA sequencing libraries.
    Biotechniques 2017 Aug 1;63(2):57-64. Epub 2017 Aug 1.
    Lund University Cancer Center, Faculty of Medicine, Department of Clinical Sciences Lund, Oncology and Pathology, Lund, Sweden.
    MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Read More

    Combinational use of lipid-based reagents for efficient transfection of primary fibroblasts and hepatoblasts.
    Biotechniques 2017 Jul 1;63(1):37-39. Epub 2017 Jul 1.
    Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
    Commercially available lipid-based transfection reagents are widely used to deliver DNA to cells. However, these lipid-based transfection reagents show poor gene transfer efficiency in primary cells. Here, we demonstrate a simple method to improve gene transfer efficiency in primary fibroblasts and hepatoblasts using a combination of lipid-based transfection reagents. Read More

    Counting nuclei released from microcarrier-based cultures using pro-fluorescent nucleic acid stains and volumetric flow cytometry.
    Biotechniques 2017 Jul 1;63(1):34-36. Epub 2017 Jul 1.
    Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.
    Counting nuclei released from intact cells is a convenient and reliable approach to assess cell density during microcarrier-based culture of adherent cells. However, commonly used methods for counting nuclei, such as crystal violet staining and quantification with a hemocytometer/automated imaging system or a Coulter counter, are imprecise, laborious and, limited in throughput. Here, we describe the use of high-affinity pro-fluorescent nucleic acid stains and volumetric flow cytometry for automated counting of nuclei released from cells attached to microcarriers with improved precision and high sample throughput. Read More

    An in vitro technique to identify the RNA binding-site sequences for RNA-binding proteins.
    Biotechniques 2017 Jul 1;63(1):28-33. Epub 2017 Jul 1.
    Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, Republic of Korea.
    RNA-protein interactions play a major role in gene regulation. Although many techniques to analyze RNA-protein interactions have been developed, noteworthy challenges such as determining the RNA sequences that bind RNA-binding proteins (RBPs) remain unsolved. Here, we describe a novel technique using a 4-thio-uridine-incorporated RNA pool to identify the RBP-binding consensus sequences for RBPs produced by in vitro transcription and translation. Read More

    Template-dependent multiple displacement amplification for profiling human circulating RNA.
    Biotechniques 2017 Jul 1;63(1):21-27. Epub 2017 Jul 1.
    Division of Gastroenterology & Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO.
    Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5´ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Read More

    SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms.
    Biotechniques 2017 Jul 1;63(1):13-20. Epub 2017 Jul 1.
    GeneMill, Centre for Genomic Research, MerseyBio Building, Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
    Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. Read More

    DNA extraction for streamlined metagenomics of diverse environmental samples.
    Biotechniques 2017 Jun 1;62(6):290-293. Epub 2017 Jun 1.
    Department of Pediatrics, University of California, San Diego, La Jolla, CA.
    A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. Read More

    A minimally-invasive method for sampling human petrous bones from the cranial base for ancient DNA analysis.
    Biotechniques 2017 Jun 1;62(6):283-289. Epub 2017 Jun 1.
    School of Archaeology and Earth Institute, Belfield, University College Dublin, Ireland.
    Ancient DNA (aDNA) research involves invasive and destructive sampling procedures that are often incompatible with anthropological, anatomical, and bioarcheological analyses requiring intact skeletal remains. The osseous labyrinth inside the petrous bone has been shown to yield higher amounts of endogenous DNA than any other skeletal element; however, accessing this labyrinth in cases of a complete or reconstructed skull involves causing major structural damage to the cranial vault or base. Here, we describe a novel cranial base drilling method (CBDM) for accessing the osseous labyrinth from the cranial base that prevents damaging the surrounding cranial features, making it highly complementary to morphological analyses. Read More

    Purification of single-stranded DNA by co-polymerization with acrylamide and electrophoresis.
    Biotechniques 2017 Jun 1;62(6):275-282. Epub 2017 Jun 1.
    University of Idaho, Department of Chemistry, Moscow, ID.
    Single-stranded DNA (ssDNA) oligonucleotides are useful as aptamers, hybridization probes and for emerging applications in DNA nanotechnology. Current methods to purify ssDNA require both a strand-separation step and a separate size-separation step but may still leave double-stranded DNA (dsDNA) impurities in the sample. Here, we use commercially available acrydite DNA primers to immobilize one strand of a PCR product within a polyacrylamide matrix. Read More

    Heteroduplex cleavage assay for screening of probable zygosities resulting from CRISPR mutations in diploid single cell lines.
    Biotechniques 2017 Jun 1;62(6):268-274. Epub 2017 Jun 1.
    Advanced Analytical Technologies, Inc., Ankeny, IA.
    The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events. Read More

    Applications of optical DNA mapping in microbiology.
    Biotechniques 2017 Jun 1;62(6):255-267. Epub 2017 Jun 1.
    Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
    Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids. Read More

    Single-step PCR-based genetic sex determination of rat tissues and cells.
    Biotechniques 2017 May 1;62(5):232-233. Epub 2017 May 1.
    Institute for Reproductive Health and Regenerative Medicine, Department of Pathology & Laboratory Medicine and Department of Pediatrics, University of Kansas Medical Center, Kansas City, KS.
    The advent of genome editing strategies has expanded the range of animal models available for gene manipulation and renewed research interest in the rat. Gender is a key variable for in vivo gene function analyses. Here, we present a simple PCR-based method to determine genetic sex in the rat. Read More

    Using non-thermal irreversible electroporation to create an in vivo niche for exogenous cell engraftment.
    Biotechniques 2017 May 1;62(5):229-231. Epub 2017 May 1.
    Department of Mechanical Engineering, University of California, Berkeley, CA.
    The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo. Read More

    Characterization of a cyclic olefin polymer microcentrifuge tube.
    Biotechniques 2017 May 1;62(5):223-228. Epub 2017 May 1.
    West Pharmaceutical Services, Exton, PA.
    Here, we describe the properties of a prototype microcentrifuge tube made from the plastic cyclic olefin polymer (COP). This material has been used in the manufacture of primary containers including syringes and vials for the storage, shipment, and delivery of biotherapeutics, vaccines, and cell therapy products. Its low level of extractable substances and metals along with its glass-like clarity make COP an attractive material for the fabrication of microcentrifuge tubes and other consumable laboratory plasticware where contamination is an important consideration, such as in the storage and analysis of labile proteins, nucleic acids, and metabolites. Read More

    ESC-Track: A computer workflow for 4-D segmentation, tracking, lineage tracing and dynamic context analysis of ESCs.
    Biotechniques 2017 May 1;62(5):215-222. Epub 2017 May 1.
    Cellomics Unit.
    Embryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions. Here, we present the ESC-Track (ESC-T) workflow, which includes an automated cell and nuclear segmentation and tracking tool for 4-D (3-D + time) confocal image data sets as well as a manual editing tool for visual inspection and error correction. Read More

    A sensitive, semi-quantitative mammalian two-hybrid assay.
    Biotechniques 2017 May 1;62(5):206-214. Epub 2017 May 1.
    Department of Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Vienna, Austria.
    Protein-protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Read More

    Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.
    Biotechniques 2017 Apr 1;62(4):183-187. Epub 2017 Apr 1.
    Experimental Therapeutics Laboratory, Hanson Institute and Sansom Institute, Adelaide, Australia.
    Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Read More

    Effective DNA fragmentation technique for simple sequence repeat detection with a microsatellite-enriched library and high-throughput sequencing.
    Biotechniques 2017 Apr 1;62(4):180-182. Epub 2017 Apr 1.
    Junior College of Tokyo University of Agriculture, Tokyo, Japan.
    Two different techniques for genomic DNA fragmentation before microsatellite-enriched library construction-restriction enzyme (NlaIII and MseI) digestion and sonication-were compared to examine their effects on simple sequence repeat (SSR) detection using high-throughput sequencing. Tens of thousands of SSR regions from 5 species of the plant family Myrtaceae were detected when the output of individual samples was >1 million paired-end reads. Comparison of the two DNA fragmentation techniques showed that restriction enzyme digestion was superior to sonication for identification of heterozygous genotypes, whereas sonication was superior for detection of various SSR flanking regions with both species-specific and common characteristics. Read More

    ImageJ macros for the user-friendly analysis of soft-agar and wound-healing assays.
    Biotechniques 2017 Apr 1;62(4):175-179. Epub 2017 Apr 1.
    Laboratory of Experimental Genetics, General Biology Department - ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
    Recent advances in biological imaging techniques and the enormous amount of data they generate call for the development of computational tools for efficient and reliable high-throughput analysis. Several software applications with this functionality are available, and one of the most commonly used is ImageJ. Here, we present two independent macros (WH_NJ and SA_NJ) for automating and facilitating the analysis of images acquired from two in vitro assays frequently used in cancer studies and drug screening: the wound-healing and soft-agar assays. Read More

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