Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Fusobacterium nucleatum. (4335)

Notes: To better understand the role MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), in reducing retrotransposon activity, 293T human embryonic kidney cells expressing MOV10 and one of three retrotransposons (LINE1 (L1), Alu or SVA) were lysed in a buffer that included RNasin® Ribonuclease Inhibitor, and FLAG-tagged L1 complexes immunoprecipitated and analyzed by mass spectrometry. Several retrotransposon assays were conducted using FuGENE® HD Transfection Reagent for transfected constructs into 293T, HeLa and HeLa-HA cells. (4255)

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HEK293 cells. HEK293 cells were plated at 1 X 10e6 cells/well in 6-well plates prior to transfection. (4413)

Notes: 5-aminolevulinic acid (5-ALA)-induced protoprophyrin IX (PpIX) fluorescence is often used for diagnostics and therapy in malignant glioma surgery; however changes in the ability of malignant glioma cells to accumulate PpIX can result in erroneous interpretation of the fluorescent signal and affect the safety of fluorescence-guided surgery. Such changes can result from several factors, including drug interactions. The authors of this paper investigated the effects of antiepiletic drugs (AED) commonly given to glioma patients on the ability of glioma cells to accumulate PpIX. They looked at cell health and metabolism in glioma cells treated with these drugs, including glutathione metabolism. The GSH/GSSG-Glo™ Assay was used to generate a ratio of reduced (GSH) and oxidized (GSSG) glutathione in glioma cell lines (U-87 MG and U373 MG). Cells were seeded at 5 × 10³ cells/well in 96-well plates and treated with either 47µg/ml phenytoin, 104µg/ml levetiracetam or DMSO-only for three days, and the assay was performed as described in the technical manual. Neither test compound effected the GSH/GSSG ratio.  (4211)

Notes: The authors of this paper studied the targeting mode of the Parkinson disease-associated kinase Pink1. They constructed a number of truncation and deletion mutants in the pGEM®-4Z Vector, and verified the identity of these clones using next-generation sequencing. The authors then expressed radiolabeled versions of the various Pink1 constructs using the TNT® Coupled Rabbit Reticulocyte Lysate System. These labeled proteins were used in mitochondrial localization studies.   (4563)

Notes: The authors previously showed that there are two independent mechanisms by which Chlamydia-specific CD4 T cells clear infection in epithelial cells; an iNOS-dependent mechanism and a Plac8-dependent mechanism. To further identify the Plac8 mechanism, they used microarrays to identify a second mechanism dependent on Plac8 for terminating Chlamydia replication in epithelial cells.

Several Chlamydia-specific CD4 T cell clones were purified at the end of their culture cycle and grown for 3 days in their usual culture media plus growth factors, without Ag stimulation. Total RNA was isolated from each clone using a protocol that included an RNase-free DNase I treatment step. Specific mRNA gene reverse transcription and amplification were performed using the AccessQuick™ RT-PCR System. (4324)

Notes: These authors performed a comprehensive analysis of all Drosophila Wnt (DWnt) family members. During their study they used FuGENE^® HD reagent to transfect various constructs into Kc or S2R+ cells. They also used various firefly and Renilla luciferase reporter constructs and the GloMax^® Multi Detection system luminometer module to investigate and the ability of the DWnts to activate the canonical Wnt signaling pathway. (4198)

Notes: The 1kb DNA Ladder and 1TE, 1X, Molecular Biology Grade were used during library preparation for next-generation sequencing. (4540)

Notes: The authors used the ReliaPrep™ FFPE gDNA Miniprep System to isolate DNA from paraffin-embedded kidney sections for use in real-time PCR to detect parvovirus B19. (4235)

Notes: These authors analyzed and compared affinity-purified protein complexes from brain homogenates of wild type and huntingtin (Htt) mutant mice by mass spectrometry. Brain tissue from FLAG-tagged wild-type and Htt mice was homogenized in HEPES buffer supplemented with protease inhibitors and RNasin®. After affinity purification, protein complexes were digested using Sequencing-Grade Modified Trypsin and ProteaseMAX™ Trypsin Enhancer prior to mass-spectrometry analysis. (4227)

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)