Notes: This paper describes a method for sequencing unknown viral isolates from tissue culture using anchored random reverse transcription and PCR, pyrosequencing and data analysis. RNA was extracted from tissue culture supernatants positive for viral antigens and used in RT-PCR with random primers. Amplification products were gel-purified and used in pyrosequencing reactions. A QuantiFluor™-P Fluorometer was used to measure copy number concentration relative to a standard, prior to Roche 454 pyrosequencing. (4231)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Borrelia burgdorferi. (4315)

Notes: Genomic DNA was extracted from shoot fragments using an organic extraction procedure and purified using the Wizard® SV Gel and PCR Clean-Up System prior to template preparation by nested PCR. Products from the second PCR were separated by electrophoresis and purified using the Wizard® SV Gel and PCR Clean-Up System prior to pyrosequencing. (4548)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Acinetobacter baumannii. (4274)

Notes: In this study, the authors evaluated whether application of the phytochemical shikonin to the skin of mice was able to augment the effect of a DNA-based anti-tumor vaccine by inducing the cytokine RANTES. As part of the study, the AccessQuick™ System was used in RT-PCR analysis to determine expression of RANTES mRNA in treated and control skin samples. (4288)

Notes: The authors transiently transfected 10⁶ Vero cells using the FuGene® HD Transfection Reagent, 2µg of DNA and a reagent:DNA ratio of 6:1. (4428)

Notes: These authors investigated the role of matrix metalloproteinase (MMP-9) in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. They found that gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. The H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. They therefore investigated the role of SMYD3 overexpression on MMP-9 expression and cell migration, identifying SMYD3 as an important new regulator of MMP-9 transcription. During the study they used the GloMax® Multi Luminometer to measure luminescence and absorbance in reporter and cell viability assays. They also used the Dual-Luciferase® Reporter Assay to measure SMYD3 activity in cells transfected with a SMYD3 reporter, and the pGL4-hRluc/TK plasmid for normalization of the experimental reporter activity. GoTaq® DNA polymerase was used in semi-quantitative RT-PCR. (4189)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Desulfitobacterium hafniense. (4326)

Notes: The authors outline best practices for blood collection, processing, shipment, and storage in an effort to encourage and optimize translational research using blood from pediatric patients. The ReliaPrep^­™ Blood gDNA Miniprep System and the Maxwell^® 16 LEV Blood DNA Kit are highlighted as all-in-one methodologies for DNA purification from blood or buffy coat. (4722)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Babesia canis. (4311)

Notes: The authors examined the effect of Varicella-zoster virus (VZV) on the NF-κB pathway and found inhibition of the pathway in VZV-infected dendritic cells. To determine which open reading frame (ORF) in the virus was responsible, seven hemagglutatin-tagged ORFs were cloned and transfected into 30% confluent 293FT human embryonic kidney cells (seeded 24 hours before) using FuGENE® HD Transfection Reagent with a 2:6 DNA:reagent ratio. After 48 hours, the cells were harvested for use in flow cytometry or Western blot analysis. An NF-κB reporter assay used 293FT cells seeded in six-well plates at 30% confluency and transfected 2µg of NF-kB GFP reporter vector and 1µg of VAV ORF expression constructs with FuGENE® HD Transfection Reagent. After 24 hours, 20nM of TNF-α was added and the cells incubated another 24 hours before harvest and analysis by flow cytometry. (4245)

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect 293FT human embryonic kidney cells. Transfection conditions were as follows: Cells were grown to 30% confluency in 6-well plates prior to transfection with 3µg DNA at a 3:1 FuGENE® reagent:DNA ratio. (4415)

Notes: Swab samples from 11 species of Chinese bats were vortexed in maintenance medium, filtered through a 0.45µm pore filter, centrifuged and resuspended. Any nonencapsidated (naked) nucleic acid was digested in a cocktail of enzymes including 20U of RNase ONE™ Ribonuclease prior to DNA and RNA purification. Conserved regions of the RNA-dependent RNA polymerase gene of astroviruses were reverse transcribed, amplified and cloned into the pGEM®-T Easy Vector for sequencing. (4552)

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

Notes: The authors used a biochemical assay using Xenopus egg extracts to monitor degradation levels of two Wnt pathway components, Axin and ß-catenin, and identify modulators of the Wnt pathway. ß-catenin and Axin were expressed in vitro as firefly and Renilla luciferase fusion proteins, respectively, using the TNT^® SP6 High-Yield Protein Expression System and shown to behave in Xenopus extracts in a similar way to wildtype proteins, which were expressed as radiolabled proteins in rabbit reticulocyte lysate. Degradation of labeled proteins was monitored by SDS polyacrylamide gel electrophoresis and autoradiography, and degradation of luciferase fusion proteins was examined using the Dual-Glo^® Luciferase Assay. Using the Xenopus extract-based assay, the authors screened chemical libraries to identify two modulators of the Wnt pathway, then confirmed this inhibition in HEK 293 cells by demonstrating a decrease in ß-catenin expression with increasing concentrations of the inhibitors. This decrease in ß-catenin-Fluc levels was measured using the Steady-Glo™ Luciferase Assay, and luciferase activity was normalized to cell viability, as determined using the CellTiter-Glo^® Assay. (4181)

Notes: The ability to detect trace protease activity is important for assessing protein purification methods during process development and for confirming the absence of protease in final purified proteins. The authors describe a general protease assay using five peptide-conjugated luciferase substrates and compare it to fluorophore-conjugated general protease assays. (4156)

Notes: This paper describes the evaluation of the HDAC-Glo™ I/II and SIRT-Glo™ Assays. For all enzymes assayed (HDAC-I, HDAC-3, HDAC-6 and SIRT-1), the K_m values obtained using the luminogenic assays were comparable to those reported in the literature. Furthermore, the assays tolerated well the concentrations of DMSO used to deliver known HDAC inhibitors. The authors used the assays to generate dose-response curves and IC₅₀ values for standard inhibitors of several HDAC isoforms. They indicate that all data sets were of high quality and that the IC₅₀ values obtained were comparable to those reported in the literature. The authors screened four HDAC isoforms (HDAC-1, HDAC-3, HDAC-6 and SIRT-1) in duplicate against 640 FDA-approved drugs, and they screened HDAC-6 and SIRT-1 against the Hypha Discovery MycoDiverse natural products library. For both screens, “hits” were classified as compounds that showed >50% inhibition at their screening concentration. For the 640-drug screen, the confirmation of hits was >80%, and the false-positive hit rate was <20%. The Z´-factor values were 0.65 for the HDAC-6 screen and >0.85 for the SIRT-1 in the Hypha Discovery MycoDiverse natural products library screen. A Z´ factor of 0.5 or greater is indicative of an assay with low data variability but good dynamic range. The authors conclude that the HDAC-Glo™ I/II and SIRT-Glo™ Assays met the criteria of sensitivity, signal stability, low background, DMSO tolerance, data variability and dynamic range, and scalability required for a suitable drug-screening assay. (4137)

Notes: The authors of this paper investigated mutations affecting the hinge loop of protein kinases that appear to confer resistance to both Type I and Type II inhibitors. They introduced individual amino acid substitutions into the hinge region of six distantly related protein kinases and determined the inhibitor sensitivity of these kinases. The ADP-Glo™ Kinase Assay was used to asses the activity of the Haspin and c-Src kinases and the engineered mutants in this study. (4144)

Notes: The authors of this study evaluated the ADP-Glo™ Assay technology for use in high-throughput screening applications for inhibitors of all four known mammalian PI 4-kinases. They found that K_m values, IC₅₀ values of known inhibitors, and dose-response curves were comparable to values reported in the literature or those obtained using the standard isotopic assay. Z´-factor values for the assay in a low-volume, 384-well format were 0.72 and 0.74, indicating that the assay would be suitable for screening activities in 384- or 1536-well formats. (4129)

Notes: This paper describes the use of the ADP-Glo™ Assay technology to screen for small-molecule inhibitors of PI 4-kinases (phatidylinositol 4-kinases). The authors characterized E. coli-expressed proteins using the ADP-Glo™ Kinase Assay and saw strong signal-to-background ratios, no signal from their negative controls or the D1899A protein, and no signal in the absence of micelles. Next they compared data from the ADP-Glo™ Assay with results obtained from the standard isotopic method or results available in the literature. They were able to obtain K_m values for PI4KA and PI4KB that corresponded to published values. Finally, the authors evaluated the potential of the ADP-Glo™ Assay as a high-throughput screening tool. To assess the assay, they used Z´ factor as their statistical measure, which is a description of the dynamic range and the variability of the assay. In this test, the Z´ factor values were 0.72 and 0.74. From these results, the authors conclude that the assay would work well for screening in a 384-well system and could likely be optimized for screening in a 1536-well format. (4135)

Notes: Human Genomic DNA used as the starting material in the NGS workflow.  GoTaq® Flexi Colorless Buffer and GoTaq® Flexi Polymerase were used in amplification of template in step added to the beginning of library preparation. (4531)

Notes: DNA templates are often amplified by PCR during library generation prior to next-generation sequencing, but amplification can introduce biases and duplications that are not easily corrected. In this paper, the authors developed a simple method to count the number of input template molecules to reduce these PCR-related problems: The ligation of a degenerate base region to all fragments during library creation. To evaluate their approach to correct for biases and duplications, the authors created a library using Human Genomic DNA, amplified the library by inverse PCR using the GoTaq^® Hot Start Polymerase and 1X Colorless GoTaq^® Flexi Buffer, sequenced the resulting DNA fragments and assessed the quality of the next-generation sequencing data. (4160)

Notes: To confirm the role of a mutation in the miR-96 microRNA (miRNA) associated with an autosomal dominant hearing lost, HeLa cells (250,000 cells per well in six-well plates) were transfected with 4µg of plasmid carrying wild type or mutant miR-96 miRNA using FuGENE® HD Transfection Reagent. After 24 hours, the cells were washed and total RNA extracted. After quantitation, the RNA used in RT-PCR analysis. The entire 3´UTRs of eight putative target genes were amplified by PCR from genomic DNA and cloned into the psiCHECK™-2 Vector. HeLa cells were transiently transfected with 2µg of the 3´ UTR psiCHECK™-2 constructs and 0.2µg of a wild-type, single or double mutant miR-96 plasmid using FuGENE® HD Transfection Reagent. Forty-eight hours after transfection, the Dual-Luciferase® Reporter Assay System was used to quantify the firefly and Renilla luciferase in cell lysates. (4251)

Notes: This study characterized interactions between the proton-pumping membrane complex V-ATPase, the Arf nucleotide binding site opener ARNO, and aldolase. The authors used a combination of protein-protein interaction techniques to identify downstream effectors of ARNO and V-ATPase signaling, and identified aldolase as a specific interaction partner of ARNO that could be involved in intracellular trafficking and cytoskeletal modulation. As part of the study, the FluoroTect™ Green_Lys in vitro Translation Labeling System was used to fluorescently label recombinant proteins during in vitro translation reactions. The labeled proteins were used in pull-down experiments. (4250)

Notes: The transcription factor Nanog is required for the maintenance of embryonic stem (ES) cell pluripotency. These authors showed that the Nanog N-terminal domain is regulated by post-transcriptional modification, and that alternative splicing generates Nanog variants with different capacities for maintaining an undifferentiated cell state. As part of their study, the authors used GoScript® Reverse Transcriptase to generate cDNA from RNA extracted from cell lines expressing different Nanog variants. The cDNA was used in RT-qPCR to quantify relative expression levels. (4184)