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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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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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J. Biol. Chem. 286, 37196–206. 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-myc nuclear localization and binding to the e-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes. 2011

Ning, B., Liu., G., Liu, Y., Su, X., Anderson, G.J., Zheng, X., Chang, Y., Guo, M., Liu, Y., Zhao, Y. and Nie, G.

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)

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Blood Cells Mol. Dis. 46, 139-144. Application of MLPA assay to characterize unsolved α-globin gene rearrangements. 2011

Colosimo, A,. Gatta, V., Guida, V., Leodori, E., Foglietta, E., Rinaldi, S., Cappabianca, M.P., Amato, A., Stuppia, L., and Dallapiccola, B.

Notes: These authors used the Maxwell® 16 Blood DNA Purification Kit to isolate genomic DNA from leukocytes. (4210)

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Genome Res. 21, 1738-45. Application of microdroplet PCR for large-scale targeted bisulfite sequencing 2011

Komori, H.K., LaMere, S.A., Torkamani, A., Hart, G.T., Kotsopoulos, S., Warner, J., Samuels, M.L., Olson, J., Head, S.R., Ordoukhanian, P., Lee, P.L., Link, D.R. and Salomon, D.R.

Notes: The authors of this study sought to correlate promoter methylation with gene expression. Gene expression data was generated by RNA-seq of Jurkat cells. Amplified cDNA was prepared from total RNA, the cDNA was treated with S1 nuclease to remove single stranded nucleic acids and used as template for the sequencing libraries. (4536)

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S1 Nuclease

DNA Research 18, 93–105. The complete chloroplast genome of 17 individuals of pest species Jacobaea vulgaris: SNPs, microsatellites and barcoding markers for population and phylogenetic studies 2011

Doorduin, L., Gravendeel, B., Lammers, Y., Ariyurek, Y., Chin-A-Woeng, T. and Vrieling, K.

Notes: Wizard® SV Gel and PCR Clean-Up System was used to extract and purify DNA fragments that had been amplified by long-range PCR and separated on an agarose gel prior to next-generation sequencing on an Illumina platform. (4535)

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Hum. Mol. Genet. 21, 577–85. A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing 2011

Soldà, G., Robusto, M., Primignani, P., Castorina, P., Benzoni, E., Cesarani, A., Ambrosetti, U., Asselta, R. and Duga, S.

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)

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mBio. 2(6), e00275-11. Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system. 2011

Chen, J., Rood, J.I., and McClane, B.A.

Notes: These authors investigated whether ETX toxin production in C. perfringens type D is regulated by the Agr-like quorum sensing system, the VirS/VirR system, or both. They demonstrated that an agr mutant lacked ETX expression, and showed that lack of VirR had no effect on ETX production. The AccessQuick™ System was used in RT-PCR analysis of RNA isolated from mutant, wildtype and reconstituted (complemented) strains to confirm absence of agr transcripts in mutant strains. RT-PCR was also used to confirm the presence of etx transcripts in wild type strains, and their absence in the agr mutant. Previous studies had shown that toxin production is upregulated upon contact with enterocyte-like Caco-2 cells. This study showed that that the agr operon is required for such upregulation. (4289)

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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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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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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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PLos ONE 6(9), e24417. Metagenome plasticity of the bovine abomasal microbiota in immune animals in response to Ostertagia ostertagi infection. 2011

Li, R.W., Wu, S., Li, W., Huang, Y., and Gasbarre, L.C.

Notes: These authors performed metagenomic analysis to investigate any changes in composition of the microbial flora of the abomasa (ruminant 4th stomach compartment) in immune cattle after reinfection with the nematode Ostertagia ostertagi. DNA was extracted from abomasal contents using a QIAamp stool kit and the integrity verified using an Agilent Bioanalyzer 2000. PCR was then performed using primers targeting hypervariable regions of 16S rRNA genes. The amplified material was gel-purified and sequenced using the Roche 454 pyrosequencing system. DNA concentration was measured before and after PCR using the QuantiFluor™ Fluorometer.


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PLos ONE 6, e25263. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform 2011

Tamaki, H., Wright, C.L., Li, X., Lin, Q., Hwang, C., Wang, S., Timmapuram, J., Kamagata, Y. and Liu, W.T.

Notes: DNA was isolated from a variety of environmental samples including surface soil, drinking water biofilm, sludge from an anaerobic digester, bioreactor samples, ground water, peat soil and glacial deposit soil. The 16S rRNA gene was amplified from the DNA. PCR amplifications were run on agarose gels, and bands of the predicted sizes excised and purified using the Wizard® SV Gel and PCR Clean-Up System before pooling for pyrosequencing. (4554)

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