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Arvind Venkataraman, Mirjana Parlov, Ping Hu, Dan Schnell, Xingtao Wei, Jay P Tiesman
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. The spike-in is DNA from two organisms - Alivibrio fischeri and Rhodopseudomonas palustris, in a ratio of 4:1 added to samples before DNA extraction. With a valid workflow, the output ratio of relative abundances of these organisms should be close to 4...
September 17, 2018: BioTechniques
Ah-Mee Park, Ikuo Tsunoda
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. To obtain positive control bloody feces, we used an indomethacin-induced intestinal ulcer model in mice. By mixing small pieces of feces with a luminol solution, the fecal solution emitted visible blue-white chemiluminescence in dark field when feces contained hemoglobin...
August 24, 2018: BioTechniques
Pei-Zhong Tang, Bo Ding, Lansha Peng, Vadim Mozhayskiy, Jason Potter, Jonathan D Chesnut
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). The sensitivity level reached 0.1-10 reads-per-million  depending on the NGS platform used, which was equivalent to 0.0002-1% measured by Targeted Amplicon-seq. Application of TEG-seq was demonstrated for the evaluation of various Cas9/gRNA configurations, which suggests delivery of Cas9/gRNA ribonucleoprotein results in significantly fewer off-targets than Cas9/gRNA plasmid...
August 17, 2018: BioTechniques
Rushikesh Sable, Nithya Jambunathan, Sitanshu Singh, Sandeep Pallerla, Konstantin G Kousoulas, Seetharama Jois
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. However, most applications using this assay are for proteins expressed in the same cell. We employ PLA for the first time to study PPI of cell surface proteins on two different cells...
September 2018: BioTechniques
Takahisa Takino, Hiroshi Sato, Motoharu Seiki
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-μm2 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. After incubation, the collagen gel layers in cell strainers were easily separated and living cells in the gel were counted and analyzed...
September 2018: BioTechniques
Francesca Lake
No abstract text is available yet for this article.
September 2018: BioTechniques
Yan Cui, Lin Ma
No abstract text is available yet for this article.
September 2018: BioTechniques
Joseph Martin
The field of epigenetics has changed the way we look at both DNA and proteins. Could the same be true for RNA? Joseph Martin discusses. [Formula: see text].
September 2018: BioTechniques
Michal Koper, Seweryn Mroczek
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. We used this technique with chromatin immunoprecipitation to investigate RNA polymerase I (RNAPI) transcription activity and its rDNA gene profiles in Saccharomyces cerevisiae...
September 2018: BioTechniques
Pauline Texier, Michèle Coddeville, Patricia Bordes, Pierre Genevaux
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...
September 2018: BioTechniques
Bill Jia, Arin Wongprommoon
[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.
September 2018: BioTechniques
Ahmed E Fouda, D Maheeka Embogama, Vindya Ramanayake-Mudiyanselage, Mary Kay H Pflum
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.
September 2018: BioTechniques
Carl A Ascoli, Birte Aggeler
The benefits of polyclonal antibodies as tools for assay-specific target discovery and detection are numerous. As the future of basic research, diagnostics and biomarker discovery is dependent on high-quality reproducible data, there is a need to understand the importance and benefits of these valuable tools. All antibody forms - polyclonal, hybridoma-based monoclonal and recombinant monoclonal - have pros and cons for development, validation and use. Yet, polyclonal antibodies are embroiled in a firestorm of controversy concerning data reproducibility...
September 2018: BioTechniques
Francesca Lake
No abstract text is available yet for this article.
August 2018: BioTechniques
David Millán-Esteban, Diana Reyes-García, Zaida García-Casado, José Bañuls, José Antonio López-Guerrero, Celia Requena, Aranzazu Rodríguez-Hernández, Víctor Traves, Eduardo Nagore
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. DNA was extracted from unstained melanoma samples and wide local excision samples. Amplicon-based libraries were constructed and tested using time and quality parameters as variables...
August 2018: BioTechniques
Abigail Sawyer, Alfie Gleeson
No abstract text is available yet for this article.
August 2018: BioTechniques
Sherry J Coulter
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...
August 2018: BioTechniques
Talveen S Purba, Jack Marsh, Kayumba Ng'andu, Svitlana Kurinna, Ralf Paus
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. EU differentially incorporated across the HF epithelium. Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression...
August 2018: BioTechniques
Maanit Shapira, Olga Boico, Asi Cohen, Ruth Sagi, Ada Aharon, Roy Navon, Gali Kronenfeld, Katie Maler, Ester Pri-Or, Michal Stein, Adi Klein, Eran Eden, Kfir Oved
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. The automated protocol was validated and was able to reduce technician hands-on time by 76%, while maintaining high analytical performance...
August 2018: BioTechniques
Wataru Ikeda, Tomoya Nakatani, Akiyoshi Uemura
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. This experimental approach will aid in the elucidation of cellular and molecular dynamics in the CNS under physiological and pathological conditions...
August 2018: BioTechniques
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