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BioTechniques

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https://www.readbyqxmd.com/read/28911319/convenient-and-robust-genome-wide-targeted-expression-analysis-for-biomarker-discovery
#1
(no author information available yet)
No abstract text is available yet for this article.
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911318/development-and-optimization-of-a-high-titer-recombinant-lentivirus-system
#2
Austin Storck, James Ludtke, Leisha Kopp, Laura Juckem
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.
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911317/generation-of-ips-derived-model-cells-for-analyses-of-hair-shaft-differentiation
#3
Takumi Kido, Tomoatsu Horigome, Minori Uda, Naoki Adachi, Yohei Hirai
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)...
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911316/optimizing-t4-dna-polymerase-conditions-enhances-the-efficiency-of-one-step-sequence-and-ligation-independent-cloning
#4
Mohammad Nazrul Islam, Kyeong Won Lee, Hyung-Soon Yim, Seong Hyuk Lee, Hae Chang Jung, Jung-Hyun Lee, Jae-Yeon Jeong
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. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2...
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911315/direct-quantification-of-protein-glycan-phosphorylation
#5
Stephanie A Ketcham, Muhammad Ashraf, Chikkathur N Madhavarao
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...
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911314/construction-of-a-combinatorial-library-of-chimeric-tumor-specific-promoters
#6
Kirill N Kashkin, Igor P Chernov, Dmitry A Didych, Eugene D Sverdlov
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. The library was constructed by randomly combining promoter fragments from eight human genes involved in cell proliferation control. The pool of chimeric promoters was inserted into a lentiviral expression vector upstream of the CopGFP reporter gene, transduced into A431 cells, and enriched for active promoters by cell sorting...
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911313/-letter-to-the-editor-incorrect-assignment-of-affected-nucleotides-in-footprinting-probing-experiments
#7
Sergey Belikov, Fredrik Lackmann, Lars Wieslander
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.se or lars.wieslander@su.se.
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911312/persistent-epigenetic-memory
#8
Amber Dance
Nematodes transmit environmental information, such as temperature or food availability, across generations. Amber Dance looks at how this is possible.
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28911311/plants-in-the-crispr
#9
Sarah Webb
Sarah Webb explores how researchers are using CRISPR/Cas9 to solve agricultural problems.
September 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803546/prestige-antibodies%C3%A2-on-the-cell-atlas-from-milliporesigma
#10
(no author information available yet)
No abstract text is available yet for this article.
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803545/mass-spectrometric-characterization-of-peptides-associated-with-molecules-of-the-major-histocompatibility-complex
#11
Michael R Pisano, Michael Ford
No abstract text is available yet for this article.
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803544/overcoming-qrt-pcr-interference-by-select-carbon-nanotubes-in-assessments-of-gene-expression
#12
Sara T Humes, Shannon Hentschel, Candice M Lavelle, L Cody Smith, John A Lednicky, Navid B Saleh, Tara Sabo-Attwood
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...
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803543/bodipy-based-dye-for-no-wash-live-cell-staining-and-imaging
#13
Alexey A Pakhomov, Igor E Deyev, Natalia M Ratnikova, Stepan P Chumakov, Veronika B Mironiuk, Yuriy N Kononevich, Aziz M Muzafarov, Vladimir I Martynov
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.
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803542/spin-infection-enables-efficient-gene-delivery-to-muscle-stem-cells
#14
Yusaku Kodaka, Yoko Asakura, Atsushi Asakura
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. We compared multiple commercially available transfection reagents to determine which was optimal for retroviral infections of primary myoblasts...
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803541/ion-exchange-chromatography-purification-of-extracellular-vesicles
#15
Maja Kosanović, Bojana Milutinović, Sanja Goč, Ninoslav Mitić, Miroslava Janković
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. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans...
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803540/preparation-of-highly-multiplexed-small-rna-sequencing-libraries
#16
Helena Persson, Rolf Søkilde, Anna Chiara Pirona, Carlos Rovira
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...
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28803539/fighting-for-the-future-of-antibodies
#17
Nathan Blow
Nathan Blow looks at how efforts to create guidelines and scoring systems could change the way you buy antibodies.
August 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701148/improved-specificity-of-western-blot-following-processing-at-4%C3%A2-c-using-blotcycler
#18
(no author information available yet)
No abstract text is available yet for this article.
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701147/combinational-use-of-lipid-based-reagents-for-efficient-transfection-of-primary-fibroblasts-and-hepatoblasts
#19
Kazuhiro Ishiguro, Osamu Watanabe, Masanao Nakamura, Takeshi Yamamura, Masanobu Matsushita, Hidemi Goto, Yoshiki Hirooka
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. Our data show that combined use of Lipofectamine LTX and FuGENE HD increases the efficiency of gene transfer compared with the use of either reagent alone, and this combination achieves the best result of any pairwise combination of Lipofectamine LTX, FuGENE HD, TransFectin, and Fibroblast Transfection Reagent...
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701146/counting-nuclei-released-from-microcarrier-based-cultures-using-pro-fluorescent-nucleic-acid-stains-and-volumetric-flow-cytometry
#20
Michael Y C He, Steven A Stacker, Ralph Rossi, Michael M Halford
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...
July 1, 2017: BioTechniques
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