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Human Pathology in press. Proliferative glomerulonephritis with monoclonal immunoglobulin G3κ deposits in association with parvovirus B19 infection 2012

Fujita, E., Shimizu, A., Kaneko, T., Masuda, Y., Ishihara, C., Mii, A., Higo, S., Kajimoto, Y., Kanzaki, G., Nagasaka, S., Iino, Y., Katayama, Y. and Fukuda, Y.

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

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J. Clin. Microbiol. 50(5), 1580–5. Sensitive and rapid detection of the New Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification. 2012

Liu, W. et al.

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

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DNA Research 19, 395-406. Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer 2012

Murphy, S.J., Cheville, J.C., Zarei, S., Johnson, S.H., Sikkink, R.A., Kosari, F., Feldman, A.L., Eckloff, B.W., Karnes, R.J. and Vasmatzis, G.

Notes: Genomic rearrangements detected by NGS were mapped and confirmed by PCR. Human Genomic DNA: Male was used as a control for the confirmatory experiments. (4539)

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Cancer Res. 72, 810-820. SMYD3 Promotes Cancer Invasion by Epigenetic Upregulation of the Metalloproteinase MMP-9. 2012

Cock-Rada, A.M., Medjkane, S., Janski, N., Yousfi, N., Perichon, M., Chaussepied, M., Chluba, J., Langsley, G., and Weitzman, J.B.

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)

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J. Virol. 86, 10999–11012. Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces. 2012

Wu, Z., Ren, X., Yang, L., Hu, Y., Yang, J., He, G., Zhang, J., Dong, J., Sun, L., Du, J., Liu, L., Xue, Y., Wang, J., Yang, F., Zhang, S. and Jin, Q.

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)

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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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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. Antimicrob. Chemother. 67, 59-63. Disruption of the blaOXA-51-like gene by ISAba16 and activation of the blaOXA-58 gene leading to carbapenem resistance in Acinetobacter baumannii Ab244.

Lopes. B.S., Evans, B.A., and Amyes, S.G.B.

Notes: This study investigated the genetic basis of carbapenem resistance in the multidrug resistant Acinetobacter baumannii isolate, Ab244. Transposable elements are known to play an important role in multidrug resistance in A. baumannii. The authors used a multiplex PCR approach to detect the presence of known resistance genes and insertion elements, followed by RT-PCR to study expression of the genes identified. For RT-PCR, cDNA was synthesized from 100 ng of RNA using the AccessQuick™ RT-PCR System. Results of the study indicated that the blaOXA-132 gene was inactivated in A. Baumannii AB244 by insertion of ISAba16, and that carbapenem resistance in that isolate was due to an alternate resistance mechanism caused by overexpression of the blaOXA-58 gene. (4344)

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Nucl. Acids Res. 39, e81. A method for counting PCR template molecules with application to next-generation sequencing. 2011

Casbon, J.A., Osborne, R.J., Brenner, S. and Lichtenstein, C.P.

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)

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Cytokine 55, 79-89. GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells. 2011

Wei, W.C., Su, Y.H., Chen, S.S., Sheu, J.H., and Yang, N.S.

Notes: This study compared the effects of zymosan and LPS on human monocyte-derived dendritic cells (DCs). The  authors found that zymosan-activated DCs had a unique cytokine expression profile. In zymosan-activated DCs, high levels of GM-CSF and IL-27, rather than IL-12 p70, were involved in Th1 cell activation. As part of the study, RT-PCR was used to investigate the molecular basis of this failure to induce production of active IL-12 p70. Expression levels of the biologically inactive subunits of IL-12 p70 (p40 and p35) was assessed using the AccessQuick™ RT-PCR System. The results showed that zymosan induced expression of p40, but not p35 mRNA, indicating that lack of induction of p35 was the reason for failure to induce active IL-12 p70. (4348)

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Appl. Environ. Microbiol. 77, 2943–53. Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces. 2011

Kwan K., Cooper M., La Duc M.T., Vaishampayan P., Stam C., Benardini J.N., Scalzi G., Moissl-Eichinger C. and Venkateswaran K.

Notes: These authors used the Maxwell® 16 System to extract DNA from multiple sample collection devices containing a model microbial community (MMC) comprised of 11 distinct species of bacterial, archaeal and fungal lineages associated with spacecraft or clean-room surfaces. The authors compared cotton swabs, polyester wipes and biological sampling kits to assess the success of recovering DNA of rRNA genes for species-specific PCR analysis. (4124)

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J. Clin. Microbiol. 49(1), 335-44. Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.

Medina, R.A., et al.

Notes: These authors used extensive computational analysis of isolates from the 2009 H1N1 outbreak to identify conserved H1N1 sequence signatures that could potentially be used in diagnostic assays to track the spread of specific strains during viral outbreaks. They identified target sequences in the hemagglutinin and neuraminidase genes that were highly conserved among 2009 H1N1 isolates. They then designed primers to amplify those sequences and used them in Taqman® and SYBR® Green-based qPCR assays to create a discriminatory 2009 H1N1 detection assay. They used the AccessQuick™ System in conventional RT-PCR to first establish whether their chosen primers were specific for the 2009 H1N1 isolates. In that assay they amplified representative H1N1 strains spanning from 1930 to the 2009 H1N1, and showed that only the 2009 isolates generated product. (4284)

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Bioinformatics 27, 2027-2030. Pathogen detection using short-RNA deep sequencing subtraction and assembly 2011

Isakov, O., Modai, S. and Shomron, N.

Notes: To confirm the  Mycoplasma contamination in HIV-1 infected samples detected by high-throughput sequencing, a PCR-based Mycoplasma test was performed. Products were separated on an agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System before confirmatory sequencing. (4537)

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Sci. Signal. 4, ra51. Distinct Phosphorylation Sites on the β2-Adrenergic Receptor Establish a Barcode That Encodes Differential Functions of β-Arrestin 2011

Nobles, K.N., Xiao, K., Ahn, S., Shukla, A.K., Lam, C.M., Rajagopal, S., Strachan, R.T., Huang, T.Y., Bressler, E.A., Hara, M.R., Shenoy, S.K., Gygi, S.P. and Lefkowitz, R.J.

Notes: The authors created a stably transfected HEK293 cell line expressing a luminogenic cAMP-binding protein using GloSensor™ technology to quantify cAMP levels in live cells. The HEK293 cells express a beta2-adrenergic receptor (β2AR ), a Gs-coupled receptor, that when activated with an agonist, stimulates the production of cAMP. The cell line was used to demonstrate that the phosphorylation pattern (“barcode”) of the β2AR created by various G protein-coupled receptor kinases (GPKs) affects the binding and function of beta-arrestin and subsequent internalization of β2AR. GloSensor™ transfection and transcription were confirmed by stimulation of endogenous β2AR with isoproterenol. Endogenous β2ARs in HEK293 cells were prestimulated with either vehicle DMSO or isoproterenol, then washed and rechallenged with serially diluted isoproterenol. In cells transfected with control siRNA, pretreatment with isoproterenol induced a 50% loss of the maximal cAMP signal when rechallenged. Cells transfected with siRNA targeting GRK2, GRK6, or both GRK2 and GRK6 showed impairment of this desensitization. This GRK knockdown effect decreased the change observed in the maximal cAMP response (Emax) after isoproterenol pretreatment. (4138)

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Nucl. Acids Res. 39, e81. A method for counting PCR template molecules with application to next-generation sequencing.

J.A. Casbon, R. J. Osborne, S. Brenner and C.P. Lichtenstein

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)

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Biochem. Biophys. Res. Commun. 408, 160-166. Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells. 2011

 Yu, K.S., Jo, J.Y., Kim, S.J., Lee, Y., Bae, J.H., Chung, Y.H., and Koh, S.S.

Notes: These authors showed that expression of the serine protease inhibitor elafin is regulated by epigenetically controlled expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, resulting in reduced proliferation and increased apoptosis. Luciferase reporter assays were used to show that Foxa2 binding was required for activation of elafin expression, and that Foxa2 binding was activated by treatment with the methyltransferase inhibitor. These assays used a pGL3-Basic Vector construct in which expression of firefly luciferase was driven by the upstream region bearing the Foxa2 binding site. The pRL-TK Vector, expressing Renilla luciferase, was used as a normalization control. The AccessQuick™ System was used for RT-PCR analysis to show that Foxa2 mRNA was barely detectable in melanoma cells.  (4345)

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Acta Pharmacologica Sinica 32, 368-74. Attenuated Salmonella typhimurium carrying shRNA-expressing vectors elicit RNA interference in murine bladder tumors. 2011

Yang, N., Li, S.H., Lü, Y.Z., Chen, L.S., and Ren, D.M.

Notes: This proof-of-principle study investigated whether attenuated Salmonella typhimurium could be used as a vehicle for delivering shRNA-expressing plasmid DNA into cancer cells in mice. The authors delivered S. typhimurium bearing plasmids encoding anti-GFP shRNA orally to mice harboring tumors that expressed GFP. They were able to show that the bacteria accumulated and persisted for 40 days within the tumors, and that GFP expression in infected tumors was reduced. The AccessQuick™ RT-PCR System was used to analyze GFP expression levels in cultured cells and tumors. (4347)

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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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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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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.

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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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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PLos ONE 6, e29604. Probing the SELEX process with next-generation sequencing 2011

Schütze, T., Wilhelm, B., Greiner, N., Braun, H., Franziska, P., Möri, M., Erdman, V.A., Lehrach, H., Konthur, Z., Menger, M., Arndt, P.F. and Glökler, J.

Notes: Wizard® SV Gel and PCR Clean-Up System was used to purify products after barcodes were attached by PCR prior to sequencing on an Illumina Genome Analyzer GA2. (4553)

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Infect. Immun. 79, 2451-9. The Agr-like quorum-sensing system regulates sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A non-food-borne human gastrointestinal disease strain F5603. 2011

Li, J., Chen, J., Vidal, J.E., and  McClane, B.A.

Notes: This study explored whether the Agr-like quorum-sensing system regulates sporulation and production of the Clostridium perfringens toxins CPE and beta2 toxin. An agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, had reduced production of alpha-toxin and perfringolysin O during vegetative growth, and did not form spores efficiently. The AccessQuick™ System was used in RT-PCR analysis to confirm the presence or absence of agrB transcripts in the wild type, mutant, and complemented strains used in the study. (4546)

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Appl. Environ. Microbiol. 77, 2589–95. Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification. 2011

Han, F., Wang, F. and Ge, B.

Notes: The authors developed a loop-mediated isothermal amplification (LAMP) assay to distinguish between virulent and nonvirulent strains of Vibrio vulnificus by targeting the virulence-correlated gene (vcg). The authors performed PCR using vcg-specific primers and GoTaq® Hot Start Polymerase in parallel to confirm the LAMP results. (4163)

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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 biopsies 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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