Notes: In vivo imaging using bioluminescent reporters is a powerful tool for real-time detection of viral load and spread in an animal over time. However, construction of influenza reporter viruses is complicated because the small viral genome does not tolerate large insertions and all the viral genes are critical in vivo, making it impossible to replace any gene with a reporter. These authors describe construction of a replication-competent influenza reporter virus containing the small (19kDa), bright NanoLuc® luciferase gene. NanoLuc® luciferase activity was then used to monitor viral infection in real time in an animal model.  Bioluminescent imaging of the reporter virus allowed serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. (4435)

Notes: The authors of this study were conducting a genome-wide analysis to look for genetic loci necessary for bacterial cell growth and survival. Genomic DNA was purified from V. cholerae prior to amplification and high-throughput sequencing on the Illumina MiSeq platform. (4528)

Notes: The authors isolated initial genomic DNA from V. cholerae transposon library using the Wizard Genomic DNA Purification Kit followed ultimately by NGS with an Illumina MiSeq Instrument. (4921)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Escherichia coli. (4329)

Notes: This article describes how a miniaturized high-throughput biochemical assay for Yes1 kinase was developed and used for screening inhibitors in small molecule libraries. Kinase activity of Yes1 was assessed by dispensing 2µl of Yes1 enzyme in a 1,536-well white multiwell plate and mixing with compounds and substrate for a total kinase reaction volume of 25µl. The amount of ADP was quantitated using the ADP-Glo™ Kinase Assay and normalized to vehicle and minus-enzyme controls. Some of the inhibitors identified in the in vitro assay were tested in a cell-based assay. Two rhabdomyosarcoma cell lines, RD and RH30, were seeded at 4,000 cells/well in a 96-well plate with 100µl of culture medium. After an overnight incubation, 100µl of medium with 0, 0.1, 1, 5, 10, 15 or 20μM of inhibitor (final concentration) was added. After 48 hours, the number of cells was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (4354)

Notes:

This paper describes use of GloSenso™ technology to develop a luciferase biosensor for detection of caspase 3/7 activity. The authors stably expressed a caspase-3/7 biosensor (pGloSensor™-30F DEVDG Vector, available as a custom product from Promega) in 1833 breast cells and D54 glioma cells, which were then used to monitor caspase activation in cell culture following addition of the cell-permeable substrate (GloSensor™ cAMP Reagent). The paper demonstrates use of this  luciferase biosensor to detect apoptosis in vivo in cultured cells and in a mouse model, and also demonstrates the utility of the technology for high-throughput screening for pharmacologically active compounds. The regulation of apoptosis by caspases is used as an example in this study, biosensors to study other proteases involved in the regulation of cellular processes can be designed using the concepts described in the paper. (4525)

Notes: Total RNAs were extracted from the DRG cells using the ReliaPrep™ RNA Cell MiniPrep System and reverse transcribed. (4733)

Notes: The HaloTag^® protein tag was used in experiments to identify protein partners of KDM4A that involved in site-specific copy number gain in tumors, specifically at the 1q12h region. Expression constructs were transfected into HEK293T cells using the FuGENE^® HD Transfection Reagent. The HaloTag-KDM4A (Cat.# FHC00602) and HaloTag-Suv39h1 (Cat.# FHC09879) expression constructs were obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). Interacting proteins identified included DNA polymerase subunits and members of the minichromosome maintenance (MCM) complex. (4408)

Notes: This paper describes use of the mass-spectrometry-compatible ProteaseMax™ Surfactant to improve protein identification for in-gel digestion applications. The surfactant induced a 1.5−2 fold increase in peptide recovery from gel slices, increased sequence coverage 20−30%, and increased the number of identified proteins. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from the gel, eliminating the need for postdigestion extraction. (4434)

Notes: In this study, the authors evaluated “genotyping-by-sequencing” for the economically important lodgepole pine and white spruce conifer species. DNA samples extracted from dormant vegetative buds and DNA was quantitated using Quantifluor® ds DNA System on a SpectraFluor Plus plate-format fluorometer. (4507)

Notes: The authors extracted DNA from dormant vegetative buds and quantitated DNA using the QuantiFluor® dsDNA System on a SpectraFluor Plus plate-format fluorometer prior to NGS (4917)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Clostridium perfringens. (4319)

Notes: The authors extracted DNA from gallbladder cancer FFPE samples using the Maxwell® 16 Instrument and the FFPE Plus DNA Kit. The also performed target enrichment followed by NGS on an Illumina HiSeq 2000 Instrument. (4907)

Notes: PCR products from a tertiary TAIL-PCR were separated by agarose gel electrophoresis and purified using the Wizard® SV Gel and PCR Clean-Up Kit. Purified fragments were cloned into pGEM®-T Easy Vectors, and clones were sequenced. (4547)

Notes: These authors demonstrated use of NanoLuc® Luciferase bioluminescence for imaging applications in mice. They showed that bioluminescence could be imaged in superficial and deep tissues, and were able to monitor changes in bioluminescence over time to quantify tumor growth. Secreted NanoLuc® Luciferase was also detectable in small volumes of serum. The paper also details use of both NanoLuc® and firefly luciferase reporters in a dual assay to quantify two steps in TGFβ signaling both in intact cells and in living mice. (4439)

Notes: Rat neural stem cells were cultured in a 3D hydrogel for either 7 days or 14 days. The hydrogel was destroyed by pipeting and RNA was isolated from the cells with the ReliaPrep™ RNA Cell Miniprep System. The quantity of RNA was determined with the QuantiFluor® RNA Dye System prior to dye-based real-time amplification with the GoTaq® 1-Step RT-qPCR System. The levels of three targets were compared at the 7 day and 14 day time points. (4595)

Notes: The let-7 microRNA family are thought to act as tumor suppressors. Let-7 activity is downregulated in several cancers, and overexpression of let-7 inhibits cancer growth in some mouse models. The authors of this paper describe a sensitive luciferase-based reporter assay for detecting let-7 miRNA activity in cells. The reporter construct was based on the pmirGLO Vector, which contains firefly luciferase as the reporter gene and Renilla luciferase as an internal control. The authors inserted let-7 miRNA target sites at the 3′ end of the firefly luciferase gene. Interaction of let-7 miRNA with these target sequences resulted in reduced luciferase activity. The authors used the reporter construct to screen a kinase inhibitor library for compounds that repress let-7 activity in ovarian cancer cells, and identified GSK-3β as a potential target for therapeutics. (4406)

Notes: In this paper, the FuGENE^® HD Transfection Reagent was used to transiently transfect Vero, Hep-2 and HeLa cells. Transfection conditions were as follows: Vero cells plated in 60mm dishes at 4 × 10⁵ cells per plate and transfected with 3.5µg of DNA; Vero cells plated on 24-well plates at 5 × 10⁴ cells/well and transfected with 1µg DNA; Hep-2 cells plated in 12-well plates at 5 × 10⁴ cells/well on glass cover slips and transfected with 1µg of DNA; Hep-2 cells plated in 24-well plates at 5 × 10⁴ cells/well on glass cover slips and transfected with 0.5µg and 1µg of DNA; HeLa cells plated in 24-well plates at 5 × 10⁴ cells/well and transfected with 1µg of DNA. A 3:1 ratio of reagent to DNA was used for all transfections. (4419)

Notes: The authors extracted RNA from multiple tree species using the SV Total RNA Isolation System and performed RNA-seq using an Illumina HiSeq 2000 Instrument. (4914)

Notes: RNA was extracted from cultured A549 cells and used in RT-qPCR to analyze the effect of transfected siRNAs. (4444)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Haemophilus influenzae. (4336)

Notes: These authors evaluated three commonly used cell lysis buffers based on the number of proteins and peptides identified in shotgun UPLC-MS analysis. Detergents used included urea, SDC, and ProteaseMAX™ Surfactant. The proteins were digested with trypsin. The buffer that included ProteaseMax Surfactant gave the greatest number of peptide and protein group identifications. This increase was due to better extraction of membrane, nuclear and cytosolic fractions. (4440)

Notes: A 50µl long-range polymerase chain reaction (LRPCR) used 1.25U of GoTaq® Hot Start Polymerase to amplify 250ng of genomic DNA and generate amplicons with multiplex identifier (MID) tags. Reflex extension reactions (25µl) included 1.25U of GoTaq® Hot Start DNA Polymerase in 1X Herculase II Reaction Buffer, 0.5µl of Herculase II Fusion DNA Polymerase) and 0.3pM LRPCR products. (4550)

Notes: These authors analyzed the performance of the following reporter enzymes used to measure biological pathway modulation by small molecules: firefly luciferase, Renilla reniformis luciferase, β-lactamase, mutated forms of Renilla luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and NanoLuc® luciferase. They screened a collection of more than 42,000 PubChem compounds using purified enzyme preparations to determine hit rates and then examined structure:activity relationships. The study evaluated hit rates and inhibitor overlap between reporters. Based on these results, the authors suggest strategies to improve the construction and interpretation of assays using these reporter enzymes. (4437)

Notes: FuGENE^® HD Transfection Reagent was used to transiently transfect HeLa cells seeded on 12mm coverslips in 24-well plates. Cells were seeded at a concentration of 5 × 10⁴ cells/well and transfected after 24 hours using a 2:1 ratio of FuGENE^® HD reagent to DNA (4µl of reagent, 2µg of DNA). Transiently transfected cells were used for confocal microscopy. For luciferase assays, HEK293 cells were seeded into 48-well plates at 40% confluency, and 24 hours later, FuGENE^® HD reagent was used to transfect the cells with one of a variety of constructs. (4411)