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J. Bacteriol. 185, 1153–1160. Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp. Strain O-7. 2003

Orikoshi, H., Baba, N., Nakayama, S., Kashu, H., Miyamoto, K., Yasuda, M., Inamori, Y. and Tsujibo, H.

Notes: The SV Total RNA Isolation System was used to isolate total RNA from Alteromonas cultures. Five micrograms of the total RNA was used in reverse transcription reactions with M-MLV Reverse Transcriptase, RNase H Minus. cDNA chiB (chitinase B) products from the reaction were quantified by real-time PCR with SYBR green dye. (3023)

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J. Virol. 77, 1992-2002. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. 2003

Altmann, S.M., Mellon, M.T., Distel, D.L. and Kim, C.H.

Notes: The pGEM®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)

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J. Nanobiotechnol. 1:2. mRNA analysis of single living cells. 2003

Osada, T., Uehara, H., Kim, H. and Ikai, A.

Notes: Researchers used the Wizard® SV Gel & PCR Clean-Up System to purify rat c-fos RT-PCR products that were then quantified by A260 absorbance. These products were used as standards for quantitative PCR reactions. The researchers used an Applied Biosystems Prism 7000 sequence detection system to perform the 50μl quantitative PCR reactions. (2678)

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Nat. Biotechnol. 21(9), 1093-1097. Multicolor in vitro translation. 2003

Traverso, G., Diehl, F., Hurst, R., Shuber, A., Whitney, D., Johnson, C., Levin, B., Kinzler, K.W. and Vogelstein, B.

Notes: A region of the ORF from the APC gene from colorectal cancer and normal patients was isolated and amplified by PCR. The primers used incorporated transcription and translation sequence elements as well as N- and C-terminal FLAG and HA tags respectively. The PCR products were used as templates in TNT® T7 Quick for PCR transcription/translation reactions.  Four different colored BODIPY-fluor-labeled lysines were added to the reactions to label the proteins in the TNT® T7 Quick for PCR transcription/translation reactions. A subtractive precipitation strategy was used to purify full-length and truncated APC proteins based on the presences or absence of FLAG and HA tags. The partially purified proteins were run on SDS-PAGE gels and imaged with a Typhoon 9700 instrument to detect truncations.    (3026)

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Ann. Clin. Microbiol. Antimicrob. 2, 11. Mutations in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori isolates in Brazil. 2003

Ribeiro, M.L., Vitiello, L., Miranda, M.C., Benvengo, Y.H., Godoy, A.P., Mendonca, S. and Pedrazzoli Jr., J.

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify a 280 base-pair PCR product, which was then directly sequenced. (2791)

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J. Biol. Chem. 278, 37948–37956. NF-E2-related factor-2 mediates neuroprotection against mitochondrial complex I inhibitors and increased concentrations of intracellular calcium in primary cortical neurons. 2003

Lee, J.M., Shih, A.Y., Murphy, T.H., and Johnson, J.A.

Notes: NF-E2-related factor-2 (NRF2) upregulates transcription of antioxidant response element (ARE)-driven genes. The authors examined the sensitivity of Nrf-2/ primary mouse neurons to oxidative stress-induced apoptosis using the mitochondrial toxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPP+) and rotenone. Primary cultures were >090% neurons, as judged by immunostaining with the Anti-βIII Tubulin mAb and other neuron-specific antibodies. Neuronal cytotoxicity in the presence of MPP+ and rotenone was monitored using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay and TUNEL assays. The level of expression of the antioxidant-responsive gene quinone oxidoreductase was measured by mRNA quantitation using RT-PCR, enzyme activity assays and immunocytochemistry. The Reverse Transcription System was used to synthesize cDNA for subsequent amplification by PCR. (3570)

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Clin. Can. Res. 9, 1906-1916. Novel kidney cancer immunotherapy based on the granulocyte-macrophage colony-stimulating factor and carbonic anhydrase IX fusion gene 2003

Hernández, J.M., Bui, M.H.T., Han, K-r., Mukouyama, H., Freitas, D.G., Nguyen, D., Caliliw, R., Shintaku, P.I., Paik, S.H., Tso, C-L., Figlin, R.A., Belldegrun, A.S.

Notes: pGEM®-T Easy Vector was used to clone PCR products.  The CytoTox 96® Non-Radioactive Cytotoxicity Assay  was used to determine specific cytotoxicity of human dendritic cells that were transduced with recombinant adenoviruses containing the gene encoding a fusion protein of granulocyte-macrophage colony stimulating factor and carbonic anhydrase IX. (2674)

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J. Biol. Chem. 278 (10), 8018-8027. Nuclear factor-κB and mitogen-activated protein kinases mediate nitric oxide-enhanced transcriptional expression of interferon-kβ. 2003

Jacobs, A.T. and Ignarro, L.J.

Notes: Promega T4 Polynucleotide Kinase was used to end-label an oligo representing a NF-kB binding sequence element with [γ-32P] ATP (7,000 Ci/mmol).  Specific primers to IFN-β and IκB-α messenger RNA were used in RT-PCR to generate products for cloning into the pGEM®-T Easy Vector. The resulting plasmids were linearized with Nco I and Spe I, respectively, and used as templates for in vitro transcription using the Riboprobe® System to generate probes for use in an RNase protection assay.  The antisense probes were labeled with [α-32P]CTP (800 Ci/mmol) in the Riboprobe® reactions.  RNase ONE™ Ribonuclease was also used in this study.  (3197)

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J. Nutr. 133, 45-50. Pancreatic metallothionein-I may play a role in zinc homeostasis during maternal dietary zinc deficiency in mice 2003

Lee, D.K., Geiser, J., Dufner-Beattie, J. and Andrews, G.K.

Notes: The ImProm-II™ Reverse Transcriptase was used to amplify the protein coding region of a 2,264 base mouse MT-1 gene mRNA and GAPDH from mouse total RNA.  RT-PCR was accomplished with Pfu DNA polymerase.  The RT-PCR product was used to make a probe for Northern analysis. (2609)

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Forensic Sci. Int. 138, 116–118. Population data for 11 STR loci in northeast China Han. 2003

Zhanga, Y.J., Xub, Q.S. and Lee, J.B.

Notes: In this paper, researchers report allele frequencies for a population in Northeastern China. Data was generated using the PowerPlex® 16 System. DNA from volunteers was extracted using a phenol chloroform method. (3000)

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Forensic Sci. Int. 138, 116–8. Population data for 11 STR loci in northeast China Han. 2003

Zhang, Y.J., Xu, Q.S. and Lee, J.B.

Notes: Allele frequencies for 11 autosomal STR loci were determined in the Yan Bian region of China. Population data for nine STR loci were determined using the AmpFlSTR® Profiler Plus™ kit and for the Penta D and Penta E loci using the PowerPlex® 16 System. DNA was isolated using phenol:chloroform extraction, and 1ng was amplified as directed by the manufacturer. Amplified products were detected using an ABI PRISM® 377 DNA Sequencer. (3826)

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Int. Congr. Ser. 1239, 235–8. Portuguese population data on two pentanucleotide STR loci Penta E and Penta D. 2003

Ribeiro, T., Viriato, L., Vieira-Silva, C., Cruz, C., Espinheira, R. and Geada, H.

Notes: The authors reported population data for the Penta E and Penta D loci in 160 Portuguese Caucasians, 19 Portuguese of African origin and 150 meioses. DNA was extracted from blood samples using Chelex® resin, then purified using the Wizard® DNA Clean-Up System. Amplifications were performed using the PowerPlex® 16 System, the SGM Plus® kit and the GeneAmp® PCR System 9600. Amplified products were detected using an ABI PRISM® 377 DNA Sequencer. (3845)

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Int. Congr. Ser. 1239, 125–30. PowerPlex™ 16 analysis in the Japanese population. 2003

Hashiyada, M., Itakura, Y. and Nata, M.

Notes: The authors generated population data from 508 unrelated Japanese individuals using the PowerPlex® 16 System. DNA was collected as buccal swabs and isolated using Chelex® resin followed by phenol:chloroform extraction and ethanol precipitation. For each PowerPlex® 16 reaction, 2ng of DNA was amplified using a GeneAmp® PCR System 9700, and amplified products were detected using the ABI PRISM® 377 DNA Sequencer. (3843)

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Clin. Diagn. Lab. Immunol. 10, 339 - 344. Production of genetically engineered biotinylated interleukin-2 and its application in a rapid nonradioactive assay for T-cell activation. 2003

Jordan, R.A., Preissler, M.T., Banas, J.A. and Gosselin, E.J.

Notes: Researchers used PCR primers with Kpn I and Hind III restriction sites to clone human IL-2 from a known rhIL-2 E. coli clone containing the PTCGF-11 vector. The PCR product was digested with Kpn I and Hind III and cloned into the PinPoint™ Xa-3 Vector. Transformed E. coli JM109 clones were then pre-incubated in the presence of 8μM biotin for 2 hours before being induced with 100μM IPTG for an additional 2 hours. After induction, the cells were collected and resuspended in a lysis buffer before mechanical lysis with a French press. The lysate was then passed over a SoftLink™ Soft Release Avidin Resin column and the biotinylated rhIL-2 eluted. The resultant purified biotinylated rhIL-2 displayed similar properties and biological activity to native IL-2. Elutants from cells transformed with the PinPoint™ Xa Control Vector produced no biotinylated rhIL-2 and did not display any properties indicating that IL-2 was present. (2680)

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Biol. Reprod. 69, 1060–1068. Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. 2003

Hyun, S., Lee, G., Kim, D., Kim, .H, Lee, S., Nam, D., Jeong, Y., Kim, S., Yeom, S., Kang, S., Han, J., Lee, B. and Hwang, W.

Notes: The authors of this report developed a means of somatic cell nuclear transfer (SCNT) that led to the successful production of GFP-transfected piglets. Citing the high value of transgenic pigs in biotechnology applications, the authors conducted these studies to establish a system for the production of transgenic pigs using SCNT of GFP-transfected cells into enucleated oocytes. FuGENE® 6 Transfection Reagent was used to transfect enhanced-GFP into confluent fetal pig fibroblasts (1µg of pEGFP-N1 with 3µl of FuGENE® 6 reagent in a 35mm dish). The cells were cultured for 2–3 days until confluency and passaged once to achieve stable integration of the gene into chromosomes before use for SCNT. (4281)

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J. Cell Sci. 116, 2421-2430. PTHrP [67–86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression. 2003

Luparello, C., Sirchia, R. and Pupello, D.

Notes: Total and poly(A)+ RNA from treated and untreated 8701-BC breast cancer tumor cells were treated with RQ1 RNase-free DNase. The RNA samples were reverse transcribed using the M-MLV RNase H– point mutant reverse transcriptase and random primers to create cDNA used in downstream analyses. The cDNA from the reactions were used in PCR, differential display PCR, and semi-quantitative multiplex PCR reactions. In differential display PCR studies, the authors analyzed differentially displayed bands by staining 6% polyacrylamide gels with the SILVER SEQUENCE™ Staining Reagents. Differentially displayed bands were re-amplified and cloned using the pGEM®-T Easy Vector and high efficiency JM109 competent cells. (3020)

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Anal. Biochem. 316, 259–269. Quantitative intra-short interspersed element PCR for species-specific DNA identification. 2003

Walker, J.A., Hughes, D.A., Anders, B.A., Shewale, J., Sinha, S.K. and Batzer, M.A.

Notes: Cow (Bos taurus), horse (Equus caballus), sheep (Ovis aries), antelope (Antilocapra americana), dog (Canis familiaris), cat (Felis catus), and rabbit (Oryctolagus cuniculus) genomic DNA were obtained by extraction from tissue and blood samples using the Wizard® Genomic DNA Purification kit. SINEs (short interspersed elements) PCR was performed on various combinations of purified DNA from the above species. (3260)

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Biochem. J. 374, 359–367. Regulation of alternative splicing of Gtf2ird1and its impact on slow muscle promoter activity. 2003

Tay, E.S.E., Guven, K.L., Subramaniam, N., Polly, P., Issa, L.L., Hardeman, P.W. and Hardeman, E.C.

Notes: In this paper, ImProm-II™ Reverse Transcriptase was used to make cDNAs of MusTRD using template RNA isolated from C2C12 myotubes (C2C12) and various mouse hind limb muscle tissues. One microliter of the cDNA products from the reaction was used in real-time PCR analysis using primers for the alternately spliced MusTRD products and SYBR® Green Dye.  Data is presented as relative expression compared with the C2C12 cell line or as gel images of the resultant amplimers.  (2844)

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FEBS Lett. 554, 119-126. Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress. 2003

Becker, D., Hoth, S., Ache, P., Wenkel, S., Roelfsema, M.R.G., Meyerho, O., Hartung, W. and Hedrich, R.

Notes: M-MLV Reverse Transcriptase, RNase H Minus, was used to make first-strand cDNA from total and mRNA samples isolated from Arabidopsis seedlings and cell culture. cDNA was then diluted 1:20 in real-time PCR to quantitatively assess GORK, PP2CA, AKT2/3, KIN2 transcript levels. Data was normalized to actin transcript levels.  (3022)

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Am. J. Physiol. Renal Physiol. 284, F829–F839. Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na+/H+ exchanger. 2003

Wu, K.L., Khan, S., Lakhe-Reddy, S., Wang, L., Jarad, G., Miller, R.T., Konieczkowski, M., Brown, A.M., Sedor, J.R. and Schelling, J.R.

Notes: This paper describes amplification of rat NHE1 cDNA using primers incorporating the Xho I and Xba I restriction endonuclease sites, Kozak consensus, ATG start site, and a stop codon. The resultant 1.1 kb NHE1 encoding-PCR product was digested with Xba I and Xho I and cloned into the pTNT™ Vector.  This construct was used as a template in a TNT® T7 Quick Coupled Transcription/Translation reaction to generate substrates for an in vitro caspase-3 cleavage assay. Proteolytically cleaved fragments of NHE1 were run on a polyacrylamide gel and visualized by autoradiography. (3063)

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J. Biol. Chem. 278 (35), 33384–33391. Role of the glucocorticoid receptor for regulation of hypoxia-dependent gene expression. 2003

Kodama, T., Shimizu, N., Yoshikawa, N., Makino, Y., Ouchida, R., Okamoto, K., Hisada, T., Nakamura, H., Morimoto, C. and Tanaka, H.

Notes: The SV Total RNA Isolation System was used to isolate total RNA from HeLa cells for RT-PCR analysis. Fifty nanograms of the total RNA was used as template in AccessQuick™ RT-PCR System reactions. VEGF, ADM, GLUT3, and β-actin mRNA levels in HeLa cells under normoxia and hypoxia conditions were analyzed. (2746)

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Plant J. 36(3), 421-429. Sequence database of 1172 T-DNA insertion sites in Arabidopsis activation-tagging lines that showed phenotypes in T1 generation. 2003

Ichikawa, T., Nakazawa, M., Kawashima, M., Muto, S., Gohda, K., Suzuki, K., Ishikawa, A., Kobayashi, H., Yoshizumi, T., Tsumoto, Y., Tsuhara, Y., Iizumi, H., Goto, Y. and Matsui, M.

Notes: To find gain-of-function mutants in Arabidopsis, T-DNA insertions were screened for visible phenotypes. 1172 plants were found to have gain-of-function insertions. To map the insertion site of the T-DNA, genomic DNA was isolated on an automated workstation (Tecan Genesis®) using the Wizard® Magnetic 96 DNA Plant System The original plasmid was then isolated from the genomic DNA after digestion and transformation into bacteria. The plasmid was sequenced to determine the site of insertion. (2788)

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Proc. Natl. Acad. Sci. USA 100, 13773-13778. Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. 2003

Petryk, A., Warren, J.T., Marqués, G., Jarcho, M.P., Gilbert, L.I., Kahler, J., Parvy, J.P., Li, Y., Dauphin-Villemant, C. and O'Connor, M.B.,

Notes: Researchers used the SV Total RNA Isolation System to isolate total RNA from various tissues from Drosophila larvae. The isolated RNA from various tissues was used in reverse transcription reactions (using M-MLV Reverse Transcriptase) to make cDNAs of the shade (Shd) gene message. PCR amplification of Shd cDNA demonstrated that Shd was specifically expressed in certain Drosophila tissues. (2784)

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Mol. Pharmacol. 63(4), 915-924. Species-specific transcriptional activity of synthetic flavonoids in guinea pig and mouse cells as a result of differential activation of the aryl hydrocarbon receptor to interact with dioxin-responsive elements 2003

Zhou, J.G., Henry, E.C., Palermo, C.M., Dertinger, S.D. and Gasiewicz, T.A.

Notes: The species-specific response of mouse hepatoma (Hepa.2D) and guinea pig adenocarcinoma (GPC.16) cells to flavonoid treatment was explored in this study. Cell lines stably transfected with dioxin responsive element-driven luciferase constructs were treated with a series of flavanoids. Luciferase expression was measured using the Steady-Glo®> Luciferase Assay system. To increase the number of cells available, cells were grown on Cytodex-1 beads in a larger volume, then split into an opaque 96-well plate prior to application of the Steady-Glo® Reagent. Total RNA was isolated using the SV Total RNA Isolation System, then used in real-time PCR to quantitate luciferase and GAPDH RNA levels after treatment. (3091)

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J. Cell Sci. 115, 1643-1649. Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells. 2003

Dolci, S., Levati, L., Pellegrini, M., Faraoni, I., Graziani, G., Di Carlo, A. and Geremia, R.

Notes: RT-PCR was used to assess the response of mitotic spermatogonia to Kit1. Spermatogonia were cultured with and without Kit1 and RNA harvested after 24 hours. RT-PCR was performed in a two-step reaction using first the ImProm-II™ Reverse Transcriptase in a 20µl reaction. One microliter of the RT reaction was removed and amplified in a 50µl PCR using the PCR Master Mix. (2626)

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