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Mol. Endocrinol. Mar. 13, Epub (ahead of print). The micro-RNA miR-206 targets the human estrogen receptor-α, and represses ERα mRNA and protein expression in breast cancer cells. 2007

Adams, B.D., Furneaux, H. and White, B.

Notes: This study investigated the mechanism of silencing of the estrogen receptor α mRNA in the human breast cancer cell line, MCF-7. The authors initially used software for miRNA target prediction to analyze the 3´ UTR of the human ERα gene for potential miR-206 target sites. Two potential targets, designated hERα1 and hERα2, were identified. ERα levels were repressed in a dose-dependent manner in MCF-7 cells transfected with a synthetic pre-miR-206 duplex, and transfection of an miR-206 expression construct into MCF-7 cells also resulted in specific inhibition of ERα expression, as measured by real-time PCR and Northern blot assays. A luciferase reporter assay was then used to determine whether miR-206 interacted directly with the hERα1 and hERα2 sites in the ERα 3´UTR. Luciferase reporter constructs containing either the hERα1 or hERα2 cloned 3´ of the firefly luciferase gene showed miR-206-medisted repression of luciferase expression in HeLa cells. Mutation of the hERα1 or hERα2 sites to disrupt hybridization with the 5´ region of miR-206 restored luciferase activity, as did co-transfection with an miRNA antagonist of miR-206. Transformation of the luciferase constructs into the breast cancer cell lines, MCF-7 and MDA-MB-231, both of which expressed high levels of miR-206 as measured by real-time PCR, resulted in repression of luciferase activity. Treatment with estrogen was then shown to reduce miR-206 levels in MCF-7 cells. Luciferase assays were used to confirm this result, and levels of luciferase activity from the reporter constructs were increased upon exposure to estrogen, indicating that ERα agonists were able to decrease miR-206 levels in MCF- cells. (3603)

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Appl. Environ. Microbiol. 73, 2860-2870. The microbial community structure in petroleum-contaminated sediments corresponds to geophysical signatures. 2007

Allen, J.P., Atekwana, E.A., Atekwana, E.A., Duris, J.W., Werkema, D.D., and Rossbach, S.

Notes: These authors studied microbial community structure at various locations in an aged underground petroleum plume. DNA was purified from soil samples collected from different sites within a contaminated area. 16S rRNA genes were then amplified from the isolated DNA, and the PCR products were run on a gel and purified using the Wizard® SV Gel and PCR Clean-Up System. After subcloning into a TA vector, the 16S RNA genes were sequenced and used to identify the various Phyla represented and characterize the microbial populations present throughout the site. (3625)

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Forensic Sci. Int. 152, 89–94. Y-chromosomal STR haplotypes in a Belgian population sample and identification of a micro-variant with a flanking site mutation at DYS19. 2007

De Maesschalck, K,. Vanhoutte, E., Knaepen, K., Vanderheyden, N., Cassiman, J.J., and Decorte, R.

Notes: The authors collected DNA samples from 113 unrelated Belgian males. The PowerPlex® Y System and a GeneAmp® 9700 PCR system were used to amplify 12 Y-chromosome STR loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439). Amplification products were detected using an ABI PRISM® 3100 genetic analyzer and POP-6™ polymer. Allele and haplotype frequencies and haplotype diversity were calculated. A total of 99 different haplotypes were observed. (3655)

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Exp. Parasitol. 112, 63-65. Babesia canis vogeli: A novel PCR for its detection in dogs in Australia. 2006

Martin, A.R., Dunstan, R.H., Roberts, T.K., and Brown, G.K.

Notes: GoTaq® DNA Polymerase was used in PCR to test dog blood for the presence of Babesia canis. Genomic DNA isolated from dog blood was analyzed with primers to the variable 5’ region of the Babesia canis 18S rRNA gene. PCR was performed in 50µl reactions containing 1.25 units of GoTaq® DNA Polymerase and 10µl of GoTaq® Reaction Buffer. (3367)

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J. Biol. Chem. 281, 9963–9970. A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages. 2006

Hanington, P.C., Barreda, D.R. and Belosevic, M.

Notes: These authors expressed a recombinant form of a novel hemapoietic granulin from goldfish. This recombinant granulin was expressed with a His6 tag in a 1-liter culture of BL21 Star™ (DE3) cells and purified from the culture supernatant using the MagneHis™ Protein Purification System. The purified protein was used to immunize rabbits and produce an affinity-purified rabbit anti-goldfish granulin IgG for immunodetection. The purified protein was also added to goldfish primary kidney macrophage cultures to determine if granulin stimulates macrophage proliferation. (3567)

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J. Forensic Sci. 51, 274–281. A proposal for standardization in forensic canine DNA typing: allele nomenclature of six canine-specific STR loci. 2006

Hellmann, A.P., Rohleder, U., Eichmann, C., Pfeiffer, I., Parson, W. and Schleenbecker, U.

Notes: Saliva samples from a total of 142 dogs (36 different pure breeds and several cross breeds) were collected on cotton swabs. Genomic DNA was isolated using the ReadyAmp™ Genomic DNA Purification System. Single amplifications of six canine STR loci were performed using 1–5µl of saliva DNA extract. (3424)

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Clin. Can. Res. 12, 4515-22. Accumulation of promoter methylation suggests epigenetic progression in squamous cell carcinoma of the esophagus 2006

Guo, M., Ren, J., House, M.G., Qi, Y., Brock, M.V., Herman, J.G.

Notes: The authors studied multiple genes for aberrant DNA methylation in esophogeal cancer, to determine timing of methylation and epigenetic changes in early lesions. In addition the authors sought a role for preinvasive lesions in tumor initiation and progression. Tissues from esophageal cancers or various grades of esophogeal dysplasia were collected. A bisulfite modification was performed on genomic DNA to convert unmethylated cytosine to uracil in denatured DNA; methylated cytosines are resistant. The difference in reactivity to bisulfite treatment was used to provide unique sequences for primers to detect methylation differences. The resulting DNA samples were purified using the Wizard® DNA Clean-Up System. (4110)

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Forensic Sci. Int. 164, 75–78. Allele distribution of 15 STR loci used for human identity purposes in the Greek Cypriot population of the island of Cyprus. 2006

Cariolou, M.A., Manoli, P., Demetriou, N., Bashiardes, E., Karagrigoriou, A. and Budowle, B.

Notes: The authors generated population data for 15 autosomal STRs using the PowerPlex® 16 System and DNA samples collected from 1,475 unrelated Greek Cypriot individuals. DNA was extracted from the blood samples using organic extraction, and 0.5ng was amplified per reaction using the GeneAmp® PCR System 9700. Amplified products were analyzed using an ABI PRISM® 3100 Genetic Analyzer. (3815)

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Hum. Mol. Genet. 15, 999–1013. An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements. 2006

Disset, A., Bourgeois, C.F., Benmalek, N., Claustres, M., Stevenin, J. and Tuffery-Giraud, S.

Notes: To construct dystrophin minigenes, genomic DNA containing a mutation in dystrophin was amplified for exons 30, 31 and 32. The three PCR fragments were combined and amplified into one product. This overlap-extension PCR generated two minigenes which were then cloned into the pGEM®-T Vector and sequenced. After EcoR I digestion, the minigenes were ligated into the pSI Mammalian Expression Vector and transiently transfected into C2C12 cells for expression analysis. (3499)

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Appl. Environ. Microbiol. 72, 6070-6078. An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae. 2006

Taylor, T.V., Mitchell, T.K. and Daub, M.E.

Notes: Fungi of the genus Cercospora are plant pathogens that cause leaf spot and blight diseases, and produce the polyketide toxin cercosporin. The bacterium Xanthomonas campestris is able to rapidly degrade cercosporin. In this study, X. campestris mutants unable to degrade cercosporin were created by chemical mutagenesis. Complementation studies with a plasmid-based library of X. campestris DNA showed that the ability to degrade cercosporin was restored upon transformation with plasmids containing an oxidoreductase gene and a putative transcriptional regulator. These genes were then amplified from the mutant strains by high-fidelity PCR. The PCR products were separated by agarose gel electrophoresis, purified using the Wizard® SV Gel and PCR Clean-Up System, and subcloned into the pGEM®-T Easy Vector. The mutant genes were then sequenced to identify the nature of the mutations. (3531)

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J. Biol. Chem. 281, 17410-17419. ATP binding to a unique site in the type-1 S2- inositol 1,4,5-triphosphate receptor defines susceptibility to phosphorylation by protein kinase A. 2006

Wagner, L.E., Betzenhauser, M.J. and Yule, D.I.

Notes: N-terminal GST fusion proteins of portions of the type-1 S2- Ionsitol 1,4,5-triphosphate receptor were created using the pFN2A (GST) Flexi® Vector. The constructs were expresed in BL21 (DE3) pLysS cells and used for ATP-binding assays. (3392)

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Nucl. Acids Res. 34, 6215-6224. Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51 2006

Desfarages, S., San Filippo, J., Fournier, M. Calmels, C., Caumont-Sarcos, A., Litvak, S., Sung, P., Parissi, V.

Notes: In the process of demonstrating the role of IN in HIV-1 integration in yeast, the authors purified all DNA vectors and PCR products with the Wizard® Plus SV Miniprep System and Wizard® SV Gel System. PCR products were generated using Taq DNA Polymerase. The pGEM®-T Vector was used to clone amplification products. Sequencing was performed using BamHI, religated with T4 DNA Ligase. (3704)

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Behav. Ecol. 17, 419–429. Cichlids do not adjust reproductive skew to the availability of independent breeding options. 2006

Heg, D., Bergmuller, R., Bonfils, D., Otti, O., Bachar, A., Burri, R., Heckel, G. and Taborskya, M.

Notes: These authors sought to determine whether helpers in cooperatively breeding species reproduced or if all offspring were from the breeders. They created 32 breeding groups and tested the possibilities of reproductive skew under various conditions. Genomic DNA from breeders and helpers was isolated from ethanol-preserved 1–2 mm2 finclip samples or from whole offspring using the Wizard® Genomic DNA Purification Kit. Purified DNA was resuspended in 50µl of DNA Rehydration Solution and analyzed by PCR using seven microsatellite primer pairs. (3679)

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Appl. Environ. Microbiol. 72, 2539-2546. Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula. 2006

Mohan Nair, M.K. and Venkitanarayanan, K.S.

Notes: The outer membrane protein A (ompA) gene of Enterobacter sakazakii was amplified using PCR primers based on E. coli ompA sequences. The resulting PCR product was ligated into the pGEM®-T Easy Vector, and the sequence was confirmed. The ompA sequence was used to develop a PCR for detection of Enterobacter sakazakii in reconsituted infant formula. (3464)

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Appl. Environ. Microbiol. 72, 5463–5468. Comparative, collaborative, and on-site validation of a TaqMan® PCR method as a tool for certified production of fresh, campylobacter-free chickens. 2006

Krause, M., Josefsen, M.H., Lund, M., Jacobsen, N.R., Brorsen, L., Moos, M., Stockmarr, A. and Hoorfar, J.

Notes: To test if a real-time PCR assay would be able to detect Campylobacter spp. in various chicken samples, several laboratories were involved in a collaborative trial. Each lab was given 18 1ml samples, including 6 chicken neck skin samples, 6 shoe cover fecal samples and 6 cloacal swab samples. DNA was extracted from these samples using the MagneSil® KF, Genomic System, first tested using 20µl of paramagnetic particles (PMPs) and then using 75µl PMPs. Five microliters of purified DNA was used in real-time PCR analysis. (3558)

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J. Biol. Chem. 281, 9901–9908. CpG sites preferentially methylated by Dnmt3a in vivo. 2006

Oka, M., Rodic, N., Graddy, J., Chang, L.J. and Terada, N.

Notes: The Wizard® Genomic DNA Purification Kit was used to isolate genomic DNA from mouse embryonic stem (ES) cells, embryonic bodies (EB) and adult tissues. The purified genomic DNA was subjected to methylation-sensitive restriction fingerprinting (MSRF; digestion with methylation-sensitive restriction enzymes followed by amplification of 100ng of digested DNA), or 2µg DNA treated with bisulfite. (3419)

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Proc. Natl. Acad. Sci. USA 103, 814-819. Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis. 2006

Kim, H.J., Ryu, H., Hong, S.H., Woo, H.R., Lim, P.O., Lee, I.C., Sheen, J., Nam, H.G. and Hwang, I.

Notes: The authors characterize an Arabidopsis cytokinin-receptor mutant. RNA was isolated from leaf tissue from wildtype and transgenic Arabidopsis plants expressing various components of the cytokinin signaling pathway. The RNA was reverse transcribed using the ImProm-II™ Reverse Transcription System, and real-time PCR was performed to quantitate response regulator proteins in the signaling pathway. (3450)

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Pesticide Biochem. Physiol. 84, 236-242. Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster. 2006

Hardstone, M.C., Baker, S.A., Gao, J., Ewer, J., and Scott, J.G.

Notes: Researchers used the GoTaq® DNA Polymerase in PCR screens for excisions around a CYP6d4 gene in the HA-1829 strain of Drosophila. PCR was performed in a 20μl reaction volume using 0.4 Units of GoTaq® DNA Polymerase, 2.75mM MgCl2 and 1μl of DNA (equivalent to the DNA in approximately 1/5 to 1/10 of a fly). (3363)

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J. Appl. Microbiol. 98, 1001-1009. Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes. 2006

Dupont, K., Vogensen, F.K., and Josephsen, J.

Notes: GoTaq® DNA Polymerase was used in PCR to detect Lactococcus lactis phage DNA strains in whey samples. Phage DNA templates were amplified directly from DNase treated and boiled whey samples. For these reactions, the researchers use 0.25µl (1.25 units) of GoTaq® DNA Polymerase for each 50μl reaction. Primers were designed to distinguish various strains of Lactococcus lactis phage receptor-binding protein genes. (3362)

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J. Clin. Microbiol. 44, 3878–82. Detection of multiple noroviruses associated with an international gastroenteritis outbreak linked to oyster consumption. 2006

Le Guyader, F.S., Bon, F., DeMedici, D., Parnaudeau, S., Bertone, A., Crudeli, S., Doyle, A., Zidane, M., Suffredini, E., Kohli, E., Maddalo, F., Monini, M., Gallay, A., Pommepuy, M., Pothier, P. and Ruggeri, F.M.

Notes: The authors linked outbreaks of acute gastroenteritis in Italy and France with consumption of oysters contaminated with Norovirus. In Italy Norovirus RNA was detected in fecal samples from infected individuals using the Access RT-PCR System and primers for the Norovirus polymerase region. (3792)

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Vet. Parasitol. 135, 99-104. Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification. 2006

Theodoropoulos, G., Gazouli, M., Ikonomopoulos, J.A., Kantzoura, V., and Kominakis, A.

Notes: Researchers used GoTaq® DNA Polymerase to test sheep and goat blood samples for the presence of Babesia DNA. Primers were designed around the 18S rRNA sequence of Babesia sp. PCR was performed in a 50µl reaction volume using 1 unit of GoTaq® DNA Polymerase. Ten microliters of each amplification reaction were loaded on gels and subjected to electrophoresis. (3380)

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J. Med. Microbiol. 55, 273–277. Development of a routine laboratory direct detection system of staphylococcal enterotoxin genes. 2006

Nakayama, A., Okayama, A., Hashida, M., Yamamoto, Y., Takebe, H., Ohnaka, T., Tanaka, T. and Imai, S.

Notes: In this study, a real-time PCR assay coupled with a DNA extraction method were used to detect staphylococcal enterotoxin (SE)-encoding genes in milk, a source of staphylococcal food poisoning. Pasteurized milk prepared with known concentrations of Staphylococcus aureus was used to generate a standard curve; experimental samples were from a staphylococcal food-poisoning outbreak that occurred in Japan in June 2000. PCR inhibition was overcome by using the following DNA purification method: a sample of milk (100µl) was added to an equal volume of 0.2M sodium hydroxide and incubated at 37°C for 20 minutes. The alkaline-treated sample was neutralized using 10µl of 3M sodium acetate (pH 5.4), extracted with 1ml of petroleum ether, and centrifuged at 13,000 × g for 10 minutes at 25°C. The aqueous phase was transferred to a fresh tube and bacterial DNA was purified from the aqueous solution using the Wizard® SV Genomic DNA Purification System. (3677)

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Mol. Ecol. 15, 1405–1418. Distribution of genetic variation within and among local populations of Arabidopsis thaliana over its species range. 2006

Bakker, E.G., Stahl, E.A., Toomajian, C., Nordborg, M., Kreitman, M. and Bergelson, J.

Notes: Variation in five microsatellite loci and five SNPs covering all Arabidopsis thaliana chromosomes was examined using samples from four individuals from each of 37 local populations, including North America, England, Eastern and Western Europe, Asia, and a selection of ecotypes. Genomic DNA was extracted from leaf tissue using the Wizard® Magnetic 96 DNA Plant System and a GenoGrinder® instrument then used for amplification with the 10 loci chosen. (3427)

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Genetics 173, 1389–1395. Divergence with gene flow in Anopheles funestus from the Sudan Savanna of Burkina Faso, West Africa. 2006

Michel, A.P., Grushko, O., Guelbeogo, W.M., Lobo, N.F., Sagnon, N., Costantini, C. and Besansky, N.J.

Notes: To examine genetic variation in Anopheles funestus, genomic DNA was extracted from single mosquito carcasses (minus the abdomen) using the Wizard® SV 96 Genomic DNA Purification System on a Beckman Coulter Biomek® FX workstation. The purified genomic DNA was diluted 1:10 in water to ~5ng/µl for PCR analysis. (3415)

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Clin. Vaccine Immunol. 13, 930–935. DNA vaccine using Mycobacterium bovis Ag85B antigen induces partial protection against experimental infection in BALB/c mice. 2006

Teixeira, F.M., Teixeira, H.C., Ferreira, A.P., Rodrigues, M.F., Azevedo, V., Macedo, G.C. and Oliveira, S.C.

Notes: To test the feasibility of the Mycobacterium bovis Ag85B gene as a DNA vaccine, the gene was amplified by PCR, introducing EcoRI and XbaI restriction endonucleases onto the primers, digested with the restriction enzymes and ligated into the pCI Mammalian Expression Vector. After transformation, the constructs were isolated using the Wizard® Plus Minipreps DNA Purification System and analyzed using endonuclease digestion and DNA sequencing. To confirm production of a maltose binding protein (MBP)-Ag85B fusion protein in E. coli, the cell lysate was analyzed by Western blot, stained with a rabbit anti-MBP serum and secondary antibody Anti-Rabbit IgG (Fc), AP Conjugate (1:10,000 dilution) and developed with BCIP/NBT. BALB/c mice pretreated with 10mM cardiotoxin prior to intramuscular DNA immunization using construct pCI-Ag85B, administered at days 0, 15, 30, and 45. The immunization course was 50µl of DNA at a concentration of 1µg/µl in PBS with each animal receiving a total of 100µg of plasmid DNA. Antibody response was assessed at 15 days with an ELISA assay and cytokine presence tested two weeks after immunization using splenocytes for ELISA assays and ICC. (3552)

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