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Promega Corporation

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RNA 16, 239–50. Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression. 2010

Walters, R.W., Bradrick, S.S. and Gromeier, M.

Notes: The authors investigated the mechanism of microRNA (miRNA)-mediated regulation of both endogenous mRNAs and artificial reporter constructs. To determine whether an m7G cap and poly(A) tail are required for repression, the authors created plasmids containing eight synthetic, tandem miR-30 recognition sequences in the 3´ untranslated region (UTR) of a Renilla luciferase gene under the control of a 5´UTR that confers either cap-dependent or cap-independent translation. They used these plasmids as a template for in vitro transcription, then transfected in vitro transcripts with and without an m7G cap and poly(A) tail into 293T cells, along with miR-30 miRNA (and miR-21 as a negative control). Similar experiments were performed by transfecting 293T cell with the Renilla luciferase constructs and miR-30 and miR-21 expression vectors. Finally, the authors exchanged the artificial 3´ UTR of the Renilla luciferase construct with the 3´ UTR of BACH1, a transcription factor that is regulated by miR-155, and cotransfected 293T cells with the BACH1 3´ UTR construct and an miR-115 expression vector to examine regulation via an endogenous 3´ UTR. The Promega Primer Extension System was used to compare miR-21, miR-30 and miR-155 RNA levels in transfected and untransfected 293T cells to determine endogenous levels of these miRNAs. Renilla luciferase activity was determined using the Renilla Luciferase Assay System. The authors used Northern blot analysis and quantitative RT-PCR to determine Renilla luciferase RNA levels (and GAPDH levels for normalization purposes). The Plexor® One-Step qRT-PCR System was used for qRT-PCR. (4048)

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Antimicrob. Agents Chemother. 45, 1151–1161. Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae. 2001

Yigit, H., Queenan, A.M., Anderson, G.J., Domenech-Sanchez, A., Biddle, J.W., Steward, C.D., Alberti, S., Bush, K. and Tenover, F.C.

Notes: The study of a carbapenem resistant strain of Klebsiella pneumoniae lead to the description of a novel form of β-lactamase, which confers resistance to imipenem, meropenem, extended-spectrum cepahlosporins, and aztreonam. Total RNA used in a primer extension assay was isolated with the SV Total RNA Isolation System . Primer extension reactions were performed with Promega's Primer Extension System- AMV Reverse Transcriptase to determine the transcriptional start site of the KPC-1 β-lactamase gene. The KPC-1 β-lactamase gene was then sequenced with the fmol® DNA Sequencing System (2300)

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J. Biol. Chem. 276, 31786-31792. The Drosophila U1 and U6 gene proximal sequence elements act as important determinants of the RNA polymerase specificity of small nuclear RNA gene promoter in vitro and in vivo. 2001

McNamara-Schroeder, K.J., Hennessey, R.F., Harding, G.A., Jensen, R.C. and Stumph, W.E.

Notes: Drosophila S2 cells were transfected with a U6 maxigene plasmid and a control firefly luciferase construct (derived from the pGL2 Basic Vector) using the ProFection® Mammalian Transfection System-Calcium Phosphate. Expression of the maxigene was confirmed by isolating total RNA from an aliquot of the cells using the RNAgents® Total RNA Isolation System and performing primer extension analysis with the aid of the Primer Extension System. Luciferase assays were performed on an aliquot of cells after lysis with Reporter Lysis Buffer. (2576)

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Genetics 154, 1239-1253. The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway 2000

O'Donnell1, A. , Tiong, S., Nash, D. , Clark, D.

Notes: Polyadenylated RNA [poly(A)-RNA] was purified from 0.5 mg total RNA using the PolyATtract® mRNA Isolation System. For primer extension, a primer was radiolabeled by 5' phosphorylation with [γ-32P]ATP using the Primer Extension System. The primer extension reaction was performed using 10 µg of total RNA from adult Canton-S flies and 100 fmol of a 32P-labeled primer. Size markers were a 32P-labeled Promega phiX174/Hinf I Marker standard. (0588)

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J. Immunol. 160, 985-992. Gene organization and promoter function for CC chemokine receptor 5 (CCR5). 1998

Guignard, F., Combadiere, C., Tiffany, H.L., Murphy, P.M.

Notes: The authors used the Primer Extension System to map the transcriptional start site of human CCR5 gene. They cloned the putative CCR5 promoter, and regions thereof were cloned into the CAT in pCAT®-Basic Vector. Transient transfections were controlled for using a β-galactosidase expression plasmid, and β-galactosidase levels assayed using the β-Galactosidase Assay System. The pCAT® Vectors have been replaced by the next generation pCAT®3 Vectors. (1085)

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J. Biol. Chem. 273, 33702-33707. Genomic organization and regulation of expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) gene. 1998

Nagase, M. , Abe, J., Takahashi, K., Ando, J., Hirose, S., Fujita, T.

Notes: The Primer Extension System was used to map transcription start site. Primer extension was performed with poly-A+ RNA. The size of the extension product was 122 bases. (0641)

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J. Biol. Chem. 273, 9457-9464. Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophophoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein. 1998

Feng, J.Q., Luan, X., Wallace, J., Jing, D., Ohshima, T., Kulkarni, A.B., D'Souza, R.N., Kozak, C.A., MacDougal, M.

Notes: Reporter assays were performed in the odontoblast cell line, MO6-G3. Experimental promoter constructs were assembled in the pGL3 Basic Vector and transfections were control through the use of the pRL-SV Vector. Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. Primer extension analysis of the gene transcript was performed with the Primer Extension System. (1196)

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J. Immunol. 161, 2953-2960. Isolation, structural characterization, and chromosomal mapping of the mouse vascular adhesion protein-1 gene and promoter. 1998

Bono, P., Salmi, M., Smith, D. J., Leppanen, I., Horelli-Kuitunen, N., Palotie, A., Jalkanen, S.

Notes: The authors used Promega's AMV Primer Extension System to map the transcriptional start site of mouse VAP-1 gene. (1426)

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J. Immunol. 160, 4353-4360. Regulation of transcription of the TATA-less human complement component C4 gene. 1998

Vaishnaw, A. K. , Mitchell, T. J. , Rose, S. J. , Walport, M. J. , Morley, B. J.

Notes: The authors used the Primer Extension System - AMV reverse transcriptase to determine transcription efficiencies of transfected constructs. Also, the fmol® DNA Cycle Sequencing System was used to confirm the nature of the constructs. EMSAs were carried out using Promega's SP1 Oligonucleotides. (0212)

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J. Biol. Chem. 272, 14454-14458. Identification of a palindromic sequence that is responsible for the up- regulation of NAPDH-ferredoxin reductase in a ferredoxin I deletion strain of Azotobacter vinelandii. 1997

Yannone, S. M. , Burgess, B. K.

Notes: The pSP-luc+NF Fusion Vector was used to produce a luc+ reporter vector for use in the gram negative prokaryote, Azotobacter vinelandii. The Primer Extension System-AMV Reverse Transcriptase was used to find the transcription start site of the bacterial promoter for NAPDH-ferredoxin reductase. (0103)

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