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Am. J. Pathol. 168, 706–713. Expression of Pax2 in human renal tumor-derived endothelial cells sustains apoptosis resistance and angiogenesis. 2006

Fonsato, V., Buttiglieri, S., Deregibus, M.C., Puntorieri, V., Bussolati, B. and Camussi, G.

Notes: To further understand the transcription factor paired-box 2 (PAX2) gene, 2µg of total RNA isolated from Kaposi’s sarcoma cells were reverse transcribed, and 5µl of the RT reaction was amplified. The PCR product was then cloned into the pTARGET™ Mammalian Expression Vector and two clones identified: PAX2 in the sense orientation and a second in the antisense orientation. The clones were then transfected into two different cell lines: the antisense PAX2 into renal tumor-derived endothelial cells where PAX2 is expressed, and the sense into normal human microvascular endothelial cells where PAX2 is not normally expressed. The effects of PAX2 expression on the cell lines were then examined. (3496)

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Plant Sci. 170, 705-14. Expression profile analysis and biochemical properties of the peptide methionine sulfoxide reductase A (PMSRA) gene family in Arabidopsis. 2006

Romero, H.M., Pell, E.J. and Tien, M.

Notes: PMSRA expression was examined in 4-week old plants exposed to 10μM methyl viologen, 100μM cercosporin, photo-oxidative stress or ozone. Samples were ground in liquid nitrogen and total RNA isolated. Total RNA (2.5μg) was reverse transcribed into cDNA with random primers d(N)10, then amplified using gene-specific primers for PMSRA1—PMSRA5 and an antibody-mediated hot start containing a mixture of GoTaq® DNA Polymerase and Taq antibody (BD Biosciences, Mountain View CA). In a total volume of 25μl, the RT-PCR reaction mixture contained 2.5 units of GoTaq® DNA Polymerase, 10mM Tris–HCl (pH 8.5), 60mM KCl, 2.4mM MgCl2 and 300μM of each dNTP. For each RT-PCR, a plant 18S universal internal standard (Ambion, Austin TX) was included as a loading control. Amplification reaction conditions were as follows: 27 cycles at 94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 minute. For each analysis, three rounds of RT-PCR were conducted with three independently isolated total RNA samples. Twenty microliters from each PCR were fractionated by 1.5% (w/v) agarose gel in Tris–borate EDTA buffer and stained with 0.5% (w/v) ethidium bromide. (3370)

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Nucl. Acids Res. 34, e7. Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids. 2006

Muranaka, N., Hohsaka, T. and Sisido, M.

Notes: The authors devised an mRNA display system to generate a peptide library with multiple nonnatural amino acids incorporated into the proteins, an important feature of peptide libraries for successful drug discovery. An mRNA with 3 four-base codons at a random position was used as a template in an in vitro translation system in the presence of charged tRNAs carrying four-base codons. In vitro translations were performed using 3.6 × 1013 molecules of mRNA template and the E. coli S30 Extract System. The mRNA template contained a T7 tag sequence, so the translation products could be detected using an anti-T7 tag antibody and the Anti-Mouse IgG (H+L), AP Conjugate. The mRNA-displayed peptides also incorporated a polyhistidine tag so that they could be purified using the MagneHis™ Ni-Particles. After selecting for the desired protein characteristic, the mRNA portion of the mRNA-displayed peptides was reverse transcribed and quantitated by real-time PCR. PCR products were cloned into the pGEM®-T Vector prior to sequencing. (3651)

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Genetics 173, 2143–2149. Functional Implication of an ARG307GLY Substitution in Corticosteroid Binding Globulin, a Candidate Gene for a QTL Associated with Cortisol Variability and Obesity. 2006

Guyonnet-Duperat, V., Geverink, N., Plastow, G.S., Evans, G., Ousova, O., Croisetiere, C., Foury, A., Richard, E., Mormede, P. and Moisan, M.P.

Notes: In this study, the effects of amino acid substitutions in porcine corticosteroid-binding globulin gene (Cbg) were tested on CBG binding and affinity. Genomic DNA was isolated from whole blood of 92 female pigs studied. Cbg cDNA was obtained by reverse transcribing pig liver total RNA using M-MLV Reverse Transcriptase followed by PCR. The 1257pb cDNA PCR product was ligated into the pTARGET™ Mammalian Expression Vector. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce four different codon substitutions in the Cbg cDNA. Once created, the mutated and unmodified Cbg cDNA constructs were transfected into HEK-293T (human embryonic kidney) cells. After 48 hours, the supernatant was collected to analyze secreted CBG. (3498)

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Forensic Sci. Int. 160, 224–230. Genetic analysis of the populations from Northern and Mesopotamian provinces of Argentina by means of 15 autosomal STRs. 2006

Marino, M., Sala, A. and Corach, D.

Notes: The authors used the PowerPlex® 16 System to gather population data from 429 unrelated individuals in northern and northeastern Argentina. Blood or buccal swabs were collected, and DNA was extracted organic extraction. Fifteen autosomal STR loci were amplified using the PowerPlex® 16 System and a GeneAmp® PCR System 9600 or 9700, and amplified products were detected using an ABI PRISM® 310 or 3100-Avant Genetic Analyzer. (3820)

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Forensic Sci. Int. 160, 84–88. Genetic attributes of 15 autosomal STRs in the population of two patagonian provinces of Argentina. 2006

Marino, M., Sala, A. and Corach, D.

Notes: The authors generated population data from 913 individuals living in two patagonian provinces in Argentina using the PowerPlex® 16 System. DNA was collected as a blood or buccal swab sample, isolated using by organic extraction and amplified using the GeneAmp® PCR System 9600 or 9700. Amplification products were detected using an ABI PRISM® 310 or 3100-Avant Genetic Analyzer. (3814)

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J. Forensic Sci. 51, 709–10. Genetic polymorphism for the PowerPlex 16 system from the Chinese Tujia Ethnic Minority Group. 2006

Wei, H., Zhao, Q. and Li, S.

Notes: The authors determined allele frequency data in the Tujia population in China using the PowerPlex® 16 System. DNA was extracted from 98 whole blood samples, and 1ng was amplified using the GeneAmp® PCR System 9600. Amplified products were analyzed using the ABI PRISM® 310 Genetic Analyzer. (3774)

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Nucl. Acids Res. 34, e67. GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. 2006

Buzdin, A., Kovalskaya-Alexandrova, E., Gogvadze, E. and Sverdlov, E.

Notes: The authors selected repetitive elements in the human genome using a novel technique: GREM. T4 DNA Ligase was used to ligate adapters to digested genomic DNA prior to PCR, and exonuclease III was used to generate the necessary 5´ termini. After the final amplification, the PCR products were cloned into the pGEM®-T Vector, then sequenced. (3550)

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J. Exp. Bot. July, epub ahead of print. HaloTag™: A new versatile reporter gene system in plant cells. 2006

Lang, C., Schulze, J., Mendel, R-R. and Hänsch, R.

Notes: This paper highlights the first use of the HaloTag™ Interchangeable Protein Labeling Technology in plant cells. The cDNA for the HaloTag™ protein was amplified by PCR from the pHT2 Vector and cloned into the pGEM®-T Easy® Vector, from which it was excised and transferred to a second vector where its expression was under the control of the cauliflower mosaic virus (CaMV)-35S promoter. The construct was transformed into tobacco protoplasts and bioballistically transformed into tobacco leaf cells. Localization was followed using the HaloTag™ TMR and diAcFAM Ligands. (3503)

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Protein Expr. Purif. 47, 562–570. High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. 2006

Blommel, P.G., Martin, P.A., Wrobel, R.L., Steffen, E. and Fox, B.G.

Notes: In this study, the Flexi® Vector Systems was compared with the Gateway® Cloning System to determine its utility in high-throughput expression cloning by subcloning 96 human target genes. A direct comparison between pVP16, the Gateway® vector and the equivalent Flexi® Vector, pVP33A or K, was achieved by modifying pVP16 with the barnase gene and PmeI/SgfI restriction sites, duplicating the design available in the commercial Flexi® Vectors. Capture of genes by PCR amplification of the cDNAs was similar for both systems, but the timeline for the Flexi® Vector system was shorter at 6–8 days compared to 12 days for the Gateway® system. They also found the Flexi® Vector System was lower cost and more accurate due to the shorter primers required for the Flexi® Vector cloning. The authors found nearly twofold fewer missense errors due to the shorter amplification primers. Ninty-six cDNAs were amplified simultaneously in their protocol and PCR products were cleaned up using either the MagneSil® PCR Clean-Up System or Wizard® SV 96 PCR Clean-Up, ligated into the Flexi® Vector, and transformed into Select96™ Competent Cells. The study also compared transfer of cDNA inserts between different Flexi® Vectors and transfer of cDNA inserts between different Gateway® vectors and found similar performance in the two systems. For the Flexi® Vector test set, the authors sequenced the clones, validating the high fidelity transfer of cDNA inserts between Flexi® Vectors. (3533)

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BMC Genetics 7, 13. High resolution physical map of porcine chromosome 7 QTL region and comparative mapping of this region among vertebrate genomes. 2006

Demars, J., Riquet, J., Feve, K., Gautier, M., Morisson, M., Demeure, O., Renard, C., Chardon, P., and Milan, D.

Notes: Fifteen microliter amplification reactions performed using 0.5 Units of GoTaq® DNA Polymerase and 25 ng BAC or genomic DNA were used as templates for sequencing portions of the QTL region of porcine chromosome 7. Amplification products were verified by gel electrophoresis followed by ethidium bromide staining. (3361)

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J. Neurosci. 26, 3299-3308. Human astrocytes are resistant to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. 2006

Song, J.H., Bellail, A., Tse, M.C.L., Yong, V.W. and Hao, C.

Notes: Total RNA was extracted from human astrocytes and control A549 cells. First strand cDNA was synthesized from 3μg of total RNA using random hexamers. PCR was performed on the cDNA samples using primers for DR4, DR5, and GAPDH with GoTaq® Green Master Mix. The PCR was performed with an initial denaturation at 94°C for 2 minutes, followed by cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 45 seconds at 72°C. The PCR products were analyzed on a 1.5% agarose gel and stained with ethidium bromide. (3372)

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Stem Cells 24, 781-792. Human umbilical cord matrix stem cells: Preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. 2006

Weiss, M.L., Medicetty, S., Bledsoe, A.R., Rachakatla, R.S., Choi, M., Merchav, S., Luo, Y., Rao, M.S., Velagaleti, G. and Troyer, D.

Notes: The gene expression profile of human umbilical cord matrix stem (UCMS) cells was determined by microarray analysis and confirmed by RT-PCR. The 50 most highly expressed genes in UCMS cells tended to fall into several categories: genes associated with neurotrophic effects and morphogenesis, the three germ layers, the undifferentiated state of embryonic stem cells and extracellular adhesion molecules.The biotin dUTP-labeled cDNA probes used to query the UCMS cell microarray were synthesized using 200 units of M-MLV Reverse Transcriptase and 4µg of total RNA. (3469)

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J. Immunol. 176, 27-35. Hydrolytic and nonenzymatic functions of acetylcholinesterase comodulate hemopoietic stress responses. 2006

Grisaru, D., Pick, M., Perry, C., Sklan, E.H., Almog, R., Goldberg, I., Naparstek, E., Lessing, J.B., Soreq, H. and Deutsch, V.

Notes: Expression levels of transcription factors critical for hemopoiesis in bone marrow were determined using real-time quantitative PCR. First, total RNA was isolated from mouse bone marrow, treated with DNase I, then reverse transcribed using the ImProm-II™ Reverse Transcription System. Each reaction included 2.4µl of 25mM MgCl2, 4µl of 5X buffer, 1µl of reverse transcriptase, 1µl of dNTP mix (10mM each), 1µl of 50µM random hexamers, 0.5µl of RNasin® Ribonuclease Inhibitor (20U), and 2µl of sample RNA (200ng/µl). (3454)

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Infect. Immun. 74, 3825-3833. Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants. 2006

Shin, S.J., Wu, C-W., Steinberg, H. and Talaat, A.M.

Notes: In this study, insertional mutagenesis with the transposon TN5367 was used to generate a library of M. paratuberculosis mutants. Sequences containing transposons were then amplified, gel purified using the Wizard® SV Gel and PCR Clean-Up System, and cloned into the pGEM®-T Easy Vector prior to sequencing. Bioinformatic screening was then used to identify potential virulence determinants for further study in a mouse model of M. paratuberculosis infection. (3534)

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Cancer Res. 66, 755–762. In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor. 2006

Yu, Z., Loehr, C.V., Fischer, K.A., Louderback, M.A., Krueger, S.K., Dashwood, R.H., Kerkvliet, N.I., Pereira, C.B., Jennings-Gee, J.E., Dance, S.T., Miller, M.S., Bailey, G.S. and Williams, D.E.

Notes: To examine the role of aryl hydrocarbon receptor (AHR) in dibenzo[a,l]pyrene (DBP) tumor development, AHR-responsive and nonresponsive murine fetuses were treated in utero with DBP. Genomic DNA was isolated from lymphomas using the Wizard® Genomic DNA Purification Kit. Two microliters of the purified DNA was PCR-amplified for analysis of the Ki-ras locus, where mutations are associated with tumor susceptibility. (3421)

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J. Leukoc. Biol. 79, 202–13. Induction of intracellular calcium elevation by {Delta}9-tetrahydrocannabinol in T cells involves TRPC1 channels. 2006

Rao, G.K. and Kaminski, N.E.

Notes: The authors studied the relationship between transient receptor potential canonical (TRPC) channels and Ca2+ elevation in the cannabinoid-2 receptor-expressing human peripheral blood-acute lymphoid leukemia (HPB-ALL) human T cell line. Total RNA from HPB-ALL cells was subjected to RT-PCR and the bands for TRPC1 were excised from a 1.2% NuSieve 3:1 agarose gel, purified using the Wizard® PCR Preps DNA Purification System and sequenced. Using 20nM synthesized siRNA specific for TRPC1 and a nonsilencing control sequence, 2.5 × 105 HPB-ALL cells/ml (a human T cell line) were transiently transfected for 48 hours using the CodeBreaker™ siRNA Transfection Reagent. After this 48-hour incubation, the siRNA-treated cells were either used for calcium determination or harvested and washed, and the RNA was isolated using the SV Total RNA Isolation System. The RNA was used for quantitative real-time PCR. (3316)

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FEBS Lett. 580, 755-762. Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). 2006

Lee, S.J., Jang, B.C., Lee, S.W., Yang, Y.I., Suh, S.I., Park, Y.M., Oh, S., Shin, J.G., Yao, S., Chen, L. and Choi, I.H.

Notes: Many cancer cells upregulate the co-signaling molecule B7-H1, confering resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System. (3451)

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J. Bacteriol. 188, 1920–1928. Involvement of the LlaKR2I methylase in expression of the AbiR bacteriophage defense system in Lactococcus lactis subsp. lactis biovar diacetylactis KR2. 2006

Yang, J.M., Deurraza, P.J., Matvienko, N. and O'Sullivan, D.J.

Notes: To avoid additional unwanted mutations introduced by a restriction enzyme deletion strategy, the Erase-a-Base® System was employed to create deletions of the LlaKR2I methylase gene, which may have a role in the abortive infection (Abi) mechanism, AbiR, that negatively affects bacteriophage DNA replication. The plasmid construct pDOU012, which contains part of the bacteriophage defense system genes, was digested with BsgI and BstEII to introduce a 3’ extension and a 5’ overhang, respectively. Exonuclease III reactions were carried out for 12 minutes at 30°C, and aliquots were taken every minute. Erythromycin-resistant transformants were selected, and deletion derivatives were identified by restriction analysis of plasmid DNA as well as by PCR and sequence analysis. Using phenotype analysis, the authors confirmed that the methylase gene was critical for expression of the AbiR phenotype. (3509)

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J. Exp. Bot. 57, 3737–46. Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. 2006

Yao, Q., Cong, L., Chang, J.L., Li, K.X., Yang, G.X. and He, G.Y.

Notes: The authors produced transgenic wheat plants with low-copy insertion of 1Ax1 and bar genes without the concomitant insertion of vector sequences using particle bombardment. Tissue-specific expression of the 1Ax1 gene, which has an endosperm-specific promoter, was examined using RT-PCR in 21 transgenic wheat lines. Total RNA was isolated from leaf, endosperm, root and inflorescence tissues, then amplified using the Access RT-PCR System. (3793)

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Cancer Res. 66, 9090-9098. MicroRNA regulates the expression of human cytochrome P450 1B1. 2006

Tsuchiya, Y., Nakajima, M., Takagi, S., Taniya, T., and Yokoi, T.

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

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J. Biol. Chem. 281, 13199-13208. Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity. 2006

Rosenkilde, M.M., Benned-Jensen, T., Andersen, H., Holst, P.J., Kledal, T.N., Luttichau, H.R., Larsen, J.K., Christensen, J.P. and Schwartz, T.W.

Notes: The expression level of the seven-transmembrane Epstein-Barr virus-induced receptor 2 (EBI2) was measured in peripheral blood mononuclear cells. Total RNA was isolated from T-lymphocytes, B-lymphocytes, monocytes and NK cells, and reverse transcribed using the ImProm-II™ Reverse Transcription System. The resulting cDNA was quantitated using real-time PCR. (3449)

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J. Physiol. 570, 283-294. Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle 2006

Payne, A.M., Zheng, Z., Messi, M.L., Milligan, C.E., González, E. and Delbono, O.

Notes: Overexpression of IGF-1 can delay or prevent aging problems in motor neurons and skeletal muscle. The authors of this paper were able to target IGF-1 to motor neurons using a fusion protein containing tetanus toxin fragment C (TTC). Motor neurons will bind, take up and transport the TTC fragment with no toxicity to the neurons. Full-length human IGF-1 cDNA was generated by PCR and inserted into the pGEM®-T Easy Vector. TTC amplified from Clostridium tetani CN655 genomic DNA was inserted into the vector. The new IFG-1-TTC insert was used for PCR to eventually produce the fusion protein for the studies. (3635)

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Int. Congr. Ser. 1288, 526–8. Multiplexing autosomal and Y-STRs loci as a powerful tool for solving old and new criminal cases. 2006

Pizzamiglio, M., Marino, A., Stabile, M. and Garofano, L.

Notes: The authors reported a partial DNA match from evidence from two robberies in Northern Italy. DNA profiles were generated with the PowerPlex® 16 System and the AmpFlSTR® Identifiler® kit, and alleles at 12 STR loci were identical. Based on the high number of matching alleles, the authors hypothesized that the two DNA donors were of the same parental lineage and generated Y-STR haplotypes, which were found to be identical. Based on the results of database searches, the authors concluded that if the number of autosomal loci with common alleles is greater than nine but not all of the loci match, there is a high chance that the DNA donors are of the same parental lineage. (3839)

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J. Biol. Chem. 281, 13915–13921. NDR2 acts as the upstream kinase of ARK5 during insulin-like growth factor-1 signaling. 2006

Suzuki, A., Ogura, T. and Esumi, H.

Notes: A deletion mutation of the serine/threonine protein kinase NDR2 was created by PCR using two mutagenesis primers and two plasmid-based primers. After amplification, the two products were run on a 1% agarose gel and extracted using the Wizard® SV Gel and PCR Clean-Up System. The purified fragments were mixed, annealed, re-amplified and then digested prior to cloning into an expression vector. The human colorectal cancer cell lines HCT-116, DLD-1, and SW480 used in the study were seeded into a 24-well plate at 5 × 104/well, and transfected using the TransFast™ Transfection Reagent. The transfection was assessed with a green fluorescent protein expression vector. (3438)

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