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Virol. J. 8, 387. First report of multiple lineages of dengue viruses type 1 in Rio de Janeiro, Brazil. 2011

dos Santos, F.B., Nogueira, F.B., Castro, M.G., Nunes, P.C., de Filippis, A.M., Fariam, N.R., Simões, J.B., Sampaio, S.A., Santos, C.R. and Nogueira, R.M.

Notes: The authors examined the strains of Dengue virus serotype 1 (DENV-1) found in the State of Rio de Janeiro since it was introduced in 1986. Viral RNA was extracted from patient serum samples or cell culture and 5µl of RNA reverse transcribed and amplified using the AccessQuick™ RT-PCR System with primers for the 2,325bp C/prM/M/E region of DENV-1. The products were sequenced and aligned to examine how the virus had evolved over time. (4342)

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Arch. Med. Sci. 7, 501-507. Frequency of Firmicutes and Bacteroidetes in gut microbiota in obese and normal weight Egyptian children and adults. 2011

Ismail, N.A., Ragab, S.H., Elbaky, A.A, Shoeib, A.R., Alhosary, Y., and Fekry, D.

Notes: These authors investigated the differences in gut microbial flora between obese and normal-weight subjects. They used the Wizard® Genomic DNA Purification Kit to extract DNA from diluted fecal extracts. The extracted DNA was analyzed by PCR to identify Bacteroidetes and Firmicutes. Differences in distribution of these phyla was between the subject groups were identified. (4220)

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Appl. Environ. Microbiol. 77, 2113–21. General suppression of Escherichia coli O157:H7 in sand-based dairy livestock bedding. 2011

Westphal, A., Williams, M.L., Baysal-Gurel, F., LeJeune, J.T. and McSpadden Gardener, B.B.

Notes: The authors investigated the suppression of E. coli O157:H7 in sand-based livestock bedding and hypothesized that suppression of E. coli O157:H7 growth was mediated by an environmentally stable population of pathogen-suppressing bacteria. These bacteria were identified by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA gene sequences isolated from used bedding followed by cloning and sequencing of the most abundant terminal restriction fragments. Amplifications were performed using the GoTaq® Flexi DNA Polymerase, then PCR products were cloned into the pGEM®-T Easy Vector. The PureYield™ Plasmid Miniprep System was used to purify plasmids for sequencing. (4165)

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Proc. Natl. Acad. Sci. USA 108, 17159–64. Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis. 2011

Crawford, M.A., Lowe, D.E., Fisher, D.J., Stibitz, S., Plaut, R.D., Beaber, J.W., Zemansky, J., Mehrad, B., Glomski, I.J., Strieter, R.M. and Hughes, M.A.

Notes: The authors identified three genetic loci involved in chemokine-mediated antimicrobial effects against Bacillus anthracis using a transposon mutant library in which a transposon is randomly inserted into the B. anthracis genome, then treating the mutant cells with chemokine to select for resistant cells. To identify the transposon insertion site, and thus the resistance-conferring loci, the authors amplified regions flanking the transposon by PCR using the GoTaq® Green Master Mix. (4167)

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PLos ONE 6(7), E22438. Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes. 2011

Racca A.C., Camolotto S.A., Ridano M.E., Bocco J.L., Genti-Raimondi S., and Panzetta-Dutari, G.M.

Notes: These authors studied KLF6 expression during human trophoblast cell differentiation, and its role in the regulation of genes associated with placental development and pregnancy maintenance. They used immunofluorescence microscopy, RT-qPCR and luciferase reporter assays to investigate cellular localization, mRNA expression, and transcriptional activation. Reporter assays were performed using various luciferase reporter constructs, the Dual-Luciferase® Assay, and the GloMax®-Multi Detection System. KLF6 was shown to play a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG. (4197)

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J. Gen. Mol. Virol. 3, 18–26. Molecular epidemiology of human enterovirus71 (HEV71) strains isolated in Peninsular Malaysia and Sabah from year 2001 to 2009 2011

Yusof, M.A., Rais, F., Abdullah, M.A., Zamri, L.A., Ali, H.M., Kassim, F.M. and Saat, Z.

Notes: This study characterized the strains of hand, foot and mouth disease (HFMD) that circulated in Peninsular Malaysia and Sabah during the last ten years. Viral RNA was extracted from vesicle, throat and rectal swabs, and 5µl of purified RNA was amplified in a 50µl reaction using the AccessQuick™ RT-PCT System with primers for VP4 or VP1. The amplified products were then sequenced and compared to known HFMD sequences. (4341)

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Proc. Natl. Acad. Sci. USA 108, 745-750. Nanoparticle-mediated delivery of siRNA targeting Parp1 extends survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells.
2011

Goldberg, M.S., Xing, D., Ren, Y., Orsulic, S., Bhatia, S.N., and Sharp, P.A.

Notes: These authors evaluated the effect of knockdown of Parp1 on survival of tumor cells after in vivo delivery of anti-Parp siRNA in a lipid-based nanoparticle format. Inhibition of Parp1 expression in mouse tumors was confirmed by qPCR analysis. Total RNA was extracted from the tumors using TRIzol® reagent (Life Technologies) and reverse transcribed using the ImProm-II™ Reverse Transcription System prior to qPCR using SYBR Green PCR Master Mix (Applied Biosystems) . (4222)

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J. Clin. Microbiol. 49, 1628–30. Novel nested direct PCR technique for malaria diagnosis using filter paper samples. 2011

Fuehrer, H.P., Fally, M.A., Habler, V.E., Starzengruber, P., Swoboda, P. and Noedl, H.

Notes: The authors developed a direct-amplification, nested PCR protocol to amplify Plasmodium DNA from 903 filter paper punches containing whole blood. The second round of nested PCR was performed using 2.5µl of PCR products from the first round and GoTaq® PCR Core System. Parallel PCRs were performed using purified DNA from blood, and both rounds of nested PCR were performed using the GoTaq® PCR Core System. (4166)

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J. Cell Sci. 124, 745–54. PERP regulates enamel formation via effects on cell-cell adhesion and gene expression. 2011

Jheon, A.H., Mostowfi, P., Snead, M.L., Ihrie, R.A., Sone, E., Pramparo, T., Attardi, L.D. and Klein, O.D.

Notes: The authors determined that PERP, a tetraspan membrane protein, is required for enamel formation during tooth development in mice. Using microarray analysis, they then identified genes that were differentially expressed in wildtype and Perp-null mice and might be involved in enamel formation. Differential expression of these genes was confirmed by qPCR using the GoTaq® qPCR Master Mix. The authors also identified an upstream regulator of Perp, P63, by transfecting cells derived from embryonic mouse teeth with a Perp-luciferase reporter construct that contained a P63 response element or mutated P63 response element. A Renilla luciferase vector was used for normalization of transfection efficiency, and luciferase assays were performed using the Dual-Luciferase® Reporter Assay System. (4168)

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Nucl. Acids Res. Dec 8, Epub ahead of print. Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts. 2011

Zhelyazkhova, P., Hammani, K., Rojas, M., Voelker, R., Vargas-Suárez, M., Börner, T., and Barkan, A.

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

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Nucl. Acids Res. [Epub ahead of print]. RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage. 2011

Dai, W., Zhang, G. and Makeyev, E.V.

Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-32P]UTP, 0.8mM Ribo m7G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of 32P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

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J. Exp. Bot. 62, 5217–31. The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes. 2011

Li, Z., Li, D., Du, X., Wang, H., Larroque, O., Jenkins, C.L., Jobling, S.A. and Morell, M.K.

Notes: The authors investigated starch synthesis in barley (Hordeum vulgare) by examining mutations in class I, class II and class III starch synthases (ssI, ssII and ssIII, respectively). Mutations of ssIIa and ssIIIa were detected by PCR using the GoTaq® Hot Start Polymerase. (4162)

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Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

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Med. Vet. Entomol. 25, 58–63. The potential of house flies to act as a vector of avian influenza subtype H5N1 under experimental conditions. 2011

Wanaratana, S., Panyim, S. and Pakpinyo, S.

Notes: To investigate if house flies could act as a vector for avian influenza virus H1N5, flies exposed to the virus were homogenated and the homogenate used to inoculate 10-day-old embryonated chicken eggs (ECEs). Allantoic fluids were collected from the eggs, RNA purified from the samples and the presence of H1N5 assessed using M-specific primers and the AccessQuick™ RT-PCR System and looking for the 276bp amplimer. (4339)

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J. Biol. Chem. 286, 19478–19488. Thrombomodulin is silenced in malignant mesothelioma by a poly(ADP-ribose) polymerase-1-mediated epigenetic mechanism. 2011

Nocchi, L., Tomasetti, M., Amati, M., Neuzil, J., Santarelli, L. and Saccucci, F.

Notes: Thrombomodulin (TM) expression was examined by isolating genomic DNA from biopies of human malignant mesothelioma and normal mesothelial tissue, and cultured cell lines with or without PARP1 silencing treated with 5-aza-2´-deoxycytidine and trichostatin alone or in combination and then subjected to biosulfide modification. To analyze methylation of TM, a CpG island in the promoter, 5´ UTR and an exon region containing 44 CpG dinucleotides were PCR amplified, cloned into the pGEM®-T Easy Vector, transformed and positive clones selected using IPTG/X-Gal and analyzed by PCR. Colonies were cultured, the plasmids isolated using the Wizard® Plus SV Minipreps DNA Purification System then 10 clones
from each sample type were sequenced. (4132)

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J. Biochem. 148, 721–32. A recombinant catalytic domain of matriptase induces detachment and apoptosis of small-intestinal epithelial IEC-6 cells cultured on laminin-coated surface. 2010

Mochida, S., Tsuzuki, S., Inouye, K. and Fushiki, T.

Notes: The authors determined that a recombinant catalytic domain of rat matriptase (His6t-S-CD) caused detachment of small-intestinal epithelial cells (IEC-6 cells) from laminin-coated plates. His6t-S-CD was expressed in the yeast P. pastoris and purified by ammonium sulfate precipitation, gel filtration through a PD-10 column, then Ni2+-chelating chromatography using HisLink™ Resin. The authors also treated IEC-6 cells with purified His6t-S-CD to determine if this domain induced apoptosis by monitoring annexin-V staining, DNA fragmentation and caspase-3 activity. For the DNA fragmentation analysis, IEC-6 cells were treated with His6t-S-CD, then harvested, and genomic DNA was purified using the Wizard® SV Gel and PCR Clean-Up System. DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide staining. (4100)

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Cancer Res. 70, 9641–9. An illegitimate microRNA target site within the 3' UTR of MDM4 affects ovarian cancer progression and chemosensitivity. 2010

Wynendaele, J., Böhnke, A., Leucci, E., Nielsen, S.J., Lambertz, I., Hammer, S., Sbrzesny, N., Kubitza, D., Wolf, A., Gradhand, E., Balschun, K., Braicu, I., Sehouli, J., Darb-Esfahani, S., Denkert, C., Thomssen, C., Hauptmann, S., Lund, A., Marine, J.C. and Bartel, F.

Notes: The authors identified a single nucleotide polymorphism (SNP) in the 3´ untranslated region (3´ UTR) of MDM4, which promotes tumorigenesis by decreasing p53 tumor suppressor function, in ovarian cancer cells. This A to C transversion creates a putative target site for the hsa-miR-191 microRNA in the MDM4-C allele, but not the wildtype MDM4-A allele. To determine if this SNP affects MDM4 translation efficiency or mRNA stability, the authors cloned a 224-bp fragment of the MDM4 3´UTRs into the psiCHECK™-2 Vector and transfected the ovarian cancer cell line A2780 with the 224A or 224C variants of the MDM4 3´ UTR. The authors used the luciferase-based psiCHECK™-2 Vector and Dual-Glo® Luciferase Assay System to show that the C variant dramatically reduces translation efficiency and/or mRNA stability. They also assessed MDM4 expression levels in A/A, A/C and C/C ovarian cancer cells and tissues using RT-qPCR. qPCR primers for MDM4 were designed using the Plexor® Primer Design Software, and assays were performed using the Plexor® qPCR System. (4157)

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J. Exp. Bot. 61, 395–404. CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis. 2010

Ruhlmann, J.M., Kram, B.W. and Carter C.J.

Notes: The authors used microarray analysis to identify genes that are differentially expressed in nectaries of Arabidopsis and may be involved in nectar synthesis and secretion. One of these genes was cell wall invertase 4 (cwinv4). The authors generated two Arabidopsis cwinv4 mutant lines to study gene function and used GoTaq® Green Master Mix to confirm the mutant genotype. Expression patterns of cwinv4 in wildtype Arabidopsis and an ortholog from Brassica rapa were examined in various tissues by RT-PCR. The reverse transcription step was performed using the Reverse Transcription System and 0.1µg of RNA. (4093)

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Am. J. Bot. 97, 1574–8. Clonal structure of wild populations and origins of horticultural stocks of Illicium parviflorum (Illiciaceae). 2010

Newell, D.L. and Morris, A.B.

Notes: The authors investigated genetic diversity in a Florida population of Illicium parviflorum, an endangered evergreen shrub, by amplifying intersimple sequence repeats (ISSRs). Amplifications were performed using GoTaq® Hot Start Polymerase. (4161)

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Genetics 184, 119–28. Detection, validation, and downstream analysis of allelic variation in gene expression. 2010

Ciobanu, D.C., Lu, L., Mozhui, K., Wang, X., Jagalur, M., Morris, J.A., Taylor, W.L., Dietz, K., Simon, P. and Williams, R.W.

Notes: Sequence variation within a gene, such as single nucleotide polymorphisms (SNPs), can lead to differences in expressions levels of corresponding mRNAs; genes that are self-regulated by this mechanism (cis modulation) are difficult to identify accurately with existing techniques. The authors used bioinformatic and molecular approaches to estimate error rates when identifying cis-modulated transcripts and developed a simple method to detect these transcripts in C57BL/6J F1 hybrid mice. This method, which they named allelic specific expression, is RT-PCR-based and uses PCR primers that flank an informative SNP to quantify the differential expression levels of transcripts. GoTaq® Flexi DNA Polymerase was used in the PCR. (4051)

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Am. J. Pathol. 177, 2347–56. Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma. 2010

de Maat, M.F., Narita, N., Benard, A., Yoshimura, T., Kuo, C., Tollenaar, R.A., de Miranda, N.F., Turner, R.R., van de Velde, C.J., Morreau, H. and Hoon, D.S.

Notes: The authors examined the methylation status of methylated-in-tumor (MINT) loci and the degree of microsatellite instability (MSI) in colorectal cancer to determine if methylation of MINT loci during the progression of adenoma to cancer was linked to MSI. They used on-slide sodium bisulfite modification and methylation-specific PCR to examine the methylation index in paraffin-embedded tissue blocks containing normal, adenoma and cancer tissues. MSI status was determined using the MSI Analysis System. Patients with instability at more than 4 markers were classified as MSI-high, and patients with instability at more than 1 marker were considered microsatellite-stable. (4106)

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J. Biomol. Scr. 15, 418–26. Epitope mapping of antibodies using a cell array-based polypeptide library. 2010

Maier, R.H., Maier, C.J., Rid, R., Hintner, H., Bauer, J.W. and Onder, K.

Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

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Appl. Environ. Microbiol. 76, 3590–9. Growth of bacteria on 3-nitropropionic acid as a sole source of carbon, nitrogen, and energy. 2010

Nishino, S.F., Shin, K.A., Payne, R.B. and Spain, J.C.

Notes: The authors identified a bacterial strain that can use the toxin 3-nitropropionic acid (3NPA) as its sole carbon and nitrogen sources and cloned the genes that encode the enzymes responsible for the initial steps in the 3NPA degradation pathway. The Pseudomonas library used to clone these genes was created using genomic DNA isolated using the Wizard® SV Genomic DNA Purification System. Closely related genes were amplified from other bacterial species for phylogenetic analysis. PCRs were performed using the GoTaq® Hot Start Polymerase. (4164)

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Clin. Can. Res. 16, 1391–401. High-resolution array comparative genomic hybridization in sporadic and celiac disease-related small bowel adenocarcinomas. 2010

Diosdado, B., Buffart, T.E., Watkins, R., Carvalho, B., Ylstra, B., Tijssen, M., Bolijn, A.S., Lewis, F., Maude, K., Verbeke, C., Nagtegaal, I.D., Grabsch, H., Mulder, C.J., Quirke, P., Howdle, P. and Meijer, G.A.

Notes: To better examine the molecular mechanisms of small bowel adenocarcinomas, DNA was extracted from paraffin-embedded tissue and tested for microsatellite instability (MSI) using the MSI Analysis System, Version 1.2. The PCR products were run on the Applied Biosystems 3130 Genetic Analyzer and analyzed using GeneScan® software. If two or more of the the BAT-25, BAT-26, NR-21, NR-24 or MONO-27 monomorphic markers had altered lengths, the tumors were designated as MSI unstable. (4112)

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J. Clin. Microbiol. 48, 3517–24. Highly sensitive and quantitative detection of the H274Y oseltamivir resistance mutation in seasonal A/H1N1 influenza virus. 2010

Operario, D.J., Moser, M.J. and St George, K.

Notes: The authors developed a RT-qPCR assay to detect the H274Y variant of the neuramidase gene from seasonal influenza A/H1N1. This variation confers resistance to oseltamivir (Tamiflu). Thus, this multiplex RT-qPCR assay can differentiate drug-resistant and wildtype A/H1N1 strains, as well as distinguish between A/H1N1 and A/H3N2, influenza B and 2009 pandemic A/H1N1 strains. Their RT-qPCR assay was based on the Plexor® One-Step qRT-PCR System. (4158)

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