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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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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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Nucl. Acids Res. 34, 2109–2116. The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription. 2006

Roberti, M., Bruni, F., Polosa, P.L., Gadaleta, M.N. and Cantatore, P.

Notes: To examine if the depletion of Drosophila transcription termination factor (DmTTF) after RNAi treatment could reduce the gene copy number, genomic DNA was isolated from RNAi-treated and untreated Drosophila embryonic D.Mel-2 cells using the Wizard® Genomic DNA Purification Kit. The mitochondrial ND3 gene and the nuclear H2B histone gene were used as probes for the Xho I-digested, Southern-blotted genomic DNA to compare the treatment groups. The Wizard® SV Gel and PCR Clean-Up System was used to clean up the PCR-amplified probes. (3418)

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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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FEBS Lett. 579, 832-8. Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways. 2006

Takami, M., Cho, E.S., Lee, S.Y., Kamijo, R. and Yim, M.

Notes: Mouse bone marrow cells and calvarial osteoblasts were cocultured for 6 days with or without 50 μM of IBMX. Total RNA was then isolated from the cells and cDNA templates prepared. cDNAs were subjected to PCR amplification with GoTaq® DNA Polymerase. Primers for mouse PDE4s, TRANCE, CTR, cathepsin K and GAPDH genes were used in this study. The PCR program was as follows: 32 (all mouse PDE4s, TRANCE, CTR, and cathepsin K) or 28 (GAPDH) cycles, after an initial denaturation step at 94°C for 3 minutes, then denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds, with a final extension at 72°C for 10 minutes. (3356)

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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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Vet. Immunol. Immunopathol. 110, 279-92. Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells. 2006

Denyer, M.S., Wileman, T.E., Stirling, C.M.A., Zuber, B., and Takamatsu, H.

Notes: In this study, GoTaq® DNA Polymerase was used in two-step RT-PCR. The ImProm-II™ Reverse Transcription System was first used to produce cDNA using an oligo d(T)15 primer. PCR was then performed using GoTaq® DNA Polymerase. Each reaction contained 2μl cDNA, 10μl GoTaq® Reaction Buffer, 1μl dNTP (10mM), 0.2μl GoTaq® DNA Polymerase, 1μl each primer (10pmol) and 34.8μl nuclease-free water. PCR was performed at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds for 35 cycles, and 72°C for 10 minutes.PCR products were visualized by agarose gel electrophoresis containing ethidium bromide and then sequenced.

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Clin. Chem. 52, 634-642. Quantification of mRNA in whole blood by assessing recovery of RNA and efficiency of cDNA synthesis. 2006

Mitsuhashi, M., Tomozawa, S., Endo, K. and Shinagawa, A.

Notes: The authors developed a method to reverse transcribe poly(A)+ RNA from leukocytes without using oligo(dT) immobilized on a 96-well plate. Four RNA targets, as well as a synthetic control RNA, were reverse transcribed using MMLV Reverse Transcriptase (1X reverse transcription buffer [50mM KCl, 10mM Tris-HCl (pH 8.3), 5.5mM MgCl2, 1nL/µL Tween 20], 1.25 mM each dNTP and 4 units of Recombinant RNasin® Ribonuclease Inhibitor), then quantitated by real-time quantitative PCR. The authors varied the concentration of MMLV Reverse Transcriptase, incubation time, and primer/template ratio] to obtain the maximum yield. Small quantities of MMLV Reverse Transcriptase were sufficient to reverse transcribe short synthetic RNA and abundant RNAs, but approximately 100 units was required for other RNAs. The synthetic control RNA was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. (3456)

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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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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. Forensic Sci. 51, 351–356. The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains. 2006

Opel, K.L., Chung, D.T., Drábek, J., Tatarek, N.E., Jantz, L.M. and McCord, B.R.

Notes: The authors developed a new set of miniplex primers for DNA typing of degraded DNA from human skeletal remains. The miniplex primers produced smaller amplicons (50–280 base pairs) than standard STR systems. The DNA-typing results obtained with the miniplex primers were compared to results obtained with the PowerPlex® 16 System. The authors determined that larger loci failed to amplify when using degraded DNA and that the degradation cut-off length of template fragments occurred predominantly at 200bp and is not kit-dependent. (3808)

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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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Mol. Cell. Biol. 17, 1331-1343. The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination. 2006

Wijeratne, A.J., Chen, C., Zhang, W., Timofejeva, L. and Ma, H.

Notes: The authors identify PARTING DANCERS as a gene involved in male meiosis in Arabidopsis using a microarray generated from meiotic-stage anthers. To confirm the sequence obtained by microarray analysis, RT-PCR was performed and the resulting cDNA was cloned into the pGEM®-T Vector and sequenced. To generate probes for in situ hybridization, fragments of ptd were amplified, cloned into the pGEM®-T Vector, then labeled with digoxygenin through in vitro transcription. (3468)

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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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Nucl. Acids Res. 34, 485-495. Nucleolin links to arsenic-induced stabilization of GADD45alpha mRNA. 2006

Zhang, Y., Bhatia, D., Xia, H., Castranova, V., Shi, X. and Chen, F.

Notes: The induction of GADD45α (growth arrest and DNA damage inducible gene 45α) in response to arsenic was examined on the protein and mRNA levels. Protein levels were determined by Western blotting; mRNA levels were determined using the AccessQuick™ RT-PCR System. Changes in GADD45α promoter activity in response to arsenic treatment were monitored in cells transiently transfected with constructs containing GADD45α promoter elements upstream of the firefly luciferase reporter gene. The Dual-Luciferase® Assay System was used to quantitate luciferase expression, and thus, promoter activity. (3440)

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Clin. Can. Res. 12, 2032-37. Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA. 2006

Wu, S., Wang, S., Wang, W., Xi, L., Tian, X., Chen, G., Wu, Y., Zhou, J., Xu, G., Lu, Y. and Ma, D.

Notes: The coding sequence of the Human Papilloma Virus (HPV16) E7 oncogene was isolated following total RNA purification from CaSki cells, RT-PCR and PCR and then cloning into the pGEM®-T Easy vector. In order to test the effectiveness of antisense HPV16 E7 therapy against cervical cancer, an adeno-associated virus vector was used to transfer the antisense construct of the E7 coding sequence into CaSki cervical cancer cells. (3395)

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Proc. Natl. Acad. Sci. USA 103, 6332-7. Nicotine inhibits apoptosis induced by chemotherapeutic drugs by up-regulating XIAP and survivin. 2006

Basgupta, P., Kinkade, R., Joshi, B., DeCook, C., Haura, E. and Chellappan, S.

Notes: RT-PCR was performed to map the subtypes of nicotinic acetylcholine receptors on A549 cells. cDNA was synthesized using the Reverse Transcription System. Northern blotting to assess XIAP and survivin expression was performed, and probes were labeled using the Prime-A-Gene® Labeling System. Apoptosis was assessed in nicotine-stimulated cells using a DeadEnd™ TUNEL Assay. (3382)

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Jpn. J. Clin. Oncol. 36, 351-356. Expression and mutation statuses of epidermal growth factor receptor in thymic epithelial tumors. 2006

Suzuki, E., Sasaki, H., Kawano, O., Endo, K., Haneda, H., Yukiue, H., Kobayashi, Y., Yano, M., and Fujii, Y.

Notes: In this study, genomic DNA was extracted from 99 frozen thymic epithelial tumor samples using the Wizard® SV Genomic DNA Purification System. Purified DNA was used in TaqMan SNP genotyping assays for 13 epidermal growth factor receptor (EGFR) gene mutations. (3585)

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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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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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Infect. Immun. 74, 3987–4001. Search for Bacillus anthracis potential vaccine candidates by a functional genomic-serologic screen. 2006

Gat, O., Grosfeld, H., Ariel, N., Inbar, I., Zaide, G., Broder, Y., Zvi, A., Chitlaru, T., Altboum, Z., Stein, D., Cohen, S. and Shafferman, A.

Notes: After bioinformatics analysis of the Bacillus anthracis genome for potential vaccine targets, the 197 ORFs were selected for amplification. Using the genomic DNA of B. anthracis strain Vollum, all PCR products were generated using a 5’ primer incorporating the T7 promoter, the Kozak sequence, unique restriction enzyme sites and a start codon and a 3’ primer with a stop codon and three more unique restriction sites. The amplicons were then in vitro transcribed and translated using the TNT® T7 Quick for PCR DNA system and radiolabeled with [35S]methionine. The products were analyzed by SDS-PAGE and autoradiography. To assess the immunoreactivity of the translated products, the proteins were immunoprecipitated using anti-B. anthracis antisera. The seropositive candidate amplified ORFs were cloned into the pCI Mammalian Expression Vector using compatible RE sites. These vectors were introduced into ICR mice using a Helios gene gun system (0.5µg plasmid DNA on 1µm gold particles) and the mice immunized in 2 week intervals. (3515)

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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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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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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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J. Endocrinol. 188, 179–192. Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary. 2006

Slot, K.A., Voorendt, M., de Boer-Brouwer, M., van Vugt, H.H. and Teerds, K.J.

Notes: The authors examined the locations and levels of apoptosis-related Fas, Fas ligand, Bcl-2, Bax and caspase-3 proteins in ovarian tissue throughout the rat estrus cycle. Protein levels were determined using Western blotting, and proteins were localized by immunhistochemistry. The presence of Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA in various ovarian tissues was monitored by RT-PCR. Reverse transcription was performed using the ImProm-II™ Reverse Transcription System, 1µg of RQ1 RNase-free DNase-treated RNA and an oligo(dT) primer. One microliter of the reverse transcription reaction was amplified by PCR, and 10µl of the amplification products were analyzed by agarose gel electrophoresis. (3724)

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