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J. Gen. Virol. 93, 2408–18. Evolution of the hepatitis E virus hypervariable region. 2012

Smith, D.B., Vanek, J., Ramalingam, S., Johannessen, I., Templeton, K. and Simmonds, P.

Notes: The authors of this study investigated the function of the hypervariable region (HVR) present in open reading frame 1 (ORF1) in the hepatitis E virus (HEV) by measuring the diversity of the HVR in HEV samples from acutely infected patients and in epidemiologically related samples. They sequenced HEV HVR PCR products from limited dilution cDNA from 8 patients PCR positive for ORF2 of HEV. HEV RNA was extracted from serum using a commercial kit, and then HEV RNA was amplified using the Access RT-PCR System. A second round of PCR was performed using GoTaq® polymerase. cDNA was generated using random hexamer or appropriate primers in the presence of Recombinant RNasin® Ribonuclease Inhibitor. (4242)

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Proc. Natl. Acad. Sci. USA 109, 13428-13433. CAG expansion induces nucleolar stress in polyglutamine diseases. 2012

Tsoi, H., Lau, T.C., Tsang, S.Y., Lau, K.F., and Chan, H.Y.

Notes: Polyglutamine diseases are neurodegenerative disorders associated with the presence of proteins containing polyglutamine repeats. These authors studied the mechanism of polygluatmine toxicity. Mutant RNAs carrying an expanded CAG repeat were shown to activate the nucleolar stress pathway and induce apoptosis. Expanded CAG RNAs were shown to interact with nucleolin, preventing it from binding to an upstream control element of the rRNA promoter and causing decreased rRNA transcription, which in turn induced apoptosis. Perturbations in rRNA transcription were identified by real-time PCR, and fluorescence in situ hybridization was used to determine that expanded CAG RNAs localized to the nucleolus. Hybridization solutions were supplemented with RNasin® Ribonuclease Inhibitor. ImProm-II™ Reverse Transcriptase was used in RT-qPCR assays. (4228)

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Nucl. Acids Res. 40, 6270–89. Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication. 2012

Bonnart C, Gérus M, Hoareau-Aveilla C, Kiss T, Caizergues-Ferrer M, Henry Y, Henras AK.

Notes: The authors of this study were investigating the relationship between ribosome biosynthesis and cell cycle progression. Specifically they were looking at the role of the protein HCA66, which is a component of the centromere, and required for centriole duplication in mammalian cells and required for nucleolar steps of 40S ribosomal subunit maturation. For studies that were conducted using HeLa cells, HeLa cell nuclear extract was prepared in the presence of Recominant RNasin® Ribonuclease Inhibitor (0.5U/µl). Prior to lysis of transfected HeLa cells for sucrose gradient experiments, cells were placed in a buffer containing Recominant RNasin® Ribonuclease Inhibitor (0.5U/µl), and for immunoprecipitation experiments involving transfected HeLa cells, cells were sonicated in the the prescence of Recominant RNasin® Ribonuclease Inhibitor (0.1U/µl). (4243)

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J. Biol. Chem. 287, 21599-21614. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis. 2012

Culver, B.P., Savas, J.N., Park, S.K., Choi, J.H., Zheng, S., Zeitlin, S.O., Yates, J.R., and Tanese, N.

Notes: These authors analyzed and compared affinity-purified protein complexes from brain homogenates of wild type and huntingtin (Htt) mutant mice by mass spectrometry. Brain tissue from FLAG-tagged wild-type and Htt mice was homogenized in HEPES buffer supplemented with protease inhibitors and RNasin®. After affinity purification, protein complexes were digested using Sequencing-Grade Modified Trypsin and ProteaseMAX™ Trypsin Enhancer prior to mass-spectrometry analysis. (4227)

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Appl. Environ. Microbiol. 78, 445–54. Responses of methanogen mcrA genes and their transcripts to an alternate dry/wet cycle of paddy field soil. 2012

Ma, K., Conrad, R. and Lu, Y.

Notes: The authors of this study investigated the microbial mechanisms associated with the reduction of methane production and emission from rice fields observed with intermittent field drainage. They looked in particular at the abundance of mcrA gene copies and transcripts from rice paddy soil fauna. The mcrA gene encodes the alpha subunit of methyl coenzyme M reductase. 

Total nucleic acid was extracted from soil samples using a phenol-chloroform procedure. For RNA analyses, DNA was hydrolyzed using RQ1 RNase-free DNase in the presence of 0.2µl Recombinant RNasin® Ribonuclease Inhibitor and then further purified using a commercial kit. cDNA synthesis was carried out using the Improm-II™ Reverse Transcription System, again in the presence of 1.0µl Recombinant RNasin® Ribonuclease Inhibitor. A clone library of transcripts was generated using the pGEM®-T Easy Vector System. The transcript standard for quantitative mcrA analysis was prepared from the in vitro transcript of a mcrA clone using the Riboprobe® in vitro Transcription Systems. (4241)

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Nucl. Acids Res. [Epub ahead of print]. RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage. 2011

Dai, W., Zhang, G. and Makeyev, E.V.

Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-32P]UTP, 0.8mM Ribo m7G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of 32P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

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J. Cell Sci. 123, 3789-3795. CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration. 2010

Galatioto, J., Mascareno, E., and Siddiqui, M.A.

Notes: These authors investigated the role of the cardiac lineage protein 1 (CLP1/HEXIM1) in skeletal myogenesis. They showed that CLP1 knockout C2C12 cells were unable to differentiate, and then investigated the hypothesis that CLP1 associates with MyoD and HDAC proteins to downregulate cell cycle genes, such as cyclin D1, and allow expression of differentiation-specific genes. RNasin® Ribonuclease inhibitor was used in coimmunoprecipitation assays investigating the interaction between CLP1 and HDAC in C2C12 cells under differentiation and non-differentiation culture conditions. RNasin® was included during cell lysate preparation prior to coimmunoprecipitation assays with antibodies directed against various HDAC proteins. The authors also performed  a luciferase reporter assay using the Dual-Luciferase® Assay System to investigate the regulation of cyclin D1 expression by CLP1, MyoD and HDAC. A luciferase reporter-cyclin D1 construct was co-transfected with MyoD, HDAC and CLP1 constructs and the effect on luciferase expression examined under differentiation and non-differentiation conditions. In differentiation medium MyoD, CLP1 and HDAC5 acted synergistically to reduce cyclin D1 expression. (4225)

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Nucl. Acids Res. 38, e167. In situ reverse transcription: the magic of strength and anonymity. 2010

Ligasová, A., and Koberna, K.

Notes: This paper describes a method for detection of polyA RNA in permeablized cells and cell sections. The method is based on incorporation of 5-bromo-2´-deoxyuridine (BrdUTP) into cDNA by AMV reverse transcriptase. The BrdUTP was easily detectable in DNA-RNA duplexes, and undetectable in DNA-DNA duplexes, making it possible to use the method to detect RNA in situ. RNasin® Ribonuclease Inhibitor was used in the reverse transcriptase reaction mix. (4226)

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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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Mol. Cell. Neurosci. 19, 447-458. AMPA Receptor-Mediated Modulation of Inward Rectifier K+ Channels in Astrocytes of Mouse Hippocampus 2002

Schroder, W., Seifert, G., Huttman, K., Hinterkeuser, S., Steinhauser, C.

Notes: The patch-clamp technique was combined with RT-PCR in acute hippocampal brain slices. After recording, each cell was harvested to perform transcript analysis and identify subunits that underlie inwardly rectifying K+ currents. The recording pipette solution was supplemented with recombinant RNasin Ribonuclease Inhibitor. After recording channel activity, cell contents were harvested through the recording pipette. RT-PCR was performed on cell contents. (2426)

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J. Bacteriol. 183, 7094-7101. Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. 2001

Vandecasteele, S.J., Peetermans, W.E., Merckx, R. and Van Eldere, J.

Notes: M-MLV Reverse Transcriptase and RNasin® Ribonuclease Inhibitor were used in a reaction to generate first strand cDNA.  The generated cDNA was used as a template for quantitative PCR in a Taqman® Assay.  The pGEM®-T Vector System was used to clone copies of each target gene so that standards could be generated.  The Wizard® Genomic DNA Purification Kit was used to isolated genomic DNA from the S. epidermidis to generate a standard curve for quantitation of genomic DNA contamination of samples. (2291)

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Am. J. Hum. Genet. 66, 790-818. Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain. 2000

Fahsold, R. , Hoffmeyer, S. , Mischung, C. , Gille, C. , Ehlers, C. , Kucukceylan, N. , Abdel-Nour, M. , Gewies, A. , Peters, H. , Kaufmann, D. , Buske, A. , Tinschert, S. , Nurnberg, P.

Notes: Reverse transcription reaction was performed in the presence of 25ug/ul Single-Stranded DNA Binding Protein and 1 unit/ul RNasin® Ribonuclease Inhibitor. PCR product and 35S-methionine were added to the TNT® Coupled Reticulocyte Lysate System to produce labeled protein in a PTT assay. (1185)

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Genetics 155, 1149-1160. RNA editing of the Drosophila para Na+ channel transcript: Evolutionary conservation and developmental regulation. 2000

Hanrahan, C.J., Palladino, M.J., Ganetsky, B. and Reenan, R.A.

Notes: Genomic DNA was extracted from 100 Drosophila melanogaster and 100 Drosophila virilis using the Wizard® Genomic DNA Purification Kit.  The isolated genomic DNA was used for PCR amplification and direct sequencing.  RT-PCR was also performed in the presence of RNasin® Ribonuclease Inhibitor and cloned amplimers were purified with the Wizard® Plus Minipreps DNA Purification System prior to fluorescent sequencing. (2505)

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Eur. J. Neurosci. 11, 617-624. Distinct population of hypothalamic dopaminergic neurons exhibit differential responses to brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). 1999

Loudes, C., Petit, F., Kordon, C., Faivre-Bauman, A.

Notes: The factors BDNF and NT-3 were used to maintain cultures. RNasin® Ribonuclease Inhibitor and Taq DNA Polymerase were used for RT-PCR of rat total RNA derived from arcuate and periventricular cultures from newborn rats as well as intermediate lobe cells from adult rats. (0745)

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Am. J. Hum. Genet. 64, 698-705. Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames 1999

Stallmeyer, B., Drugeon, G., Reiss, J. , Haenni, A. L., Mendel, R. R.

Notes: RNasin® Ribonuclease Inhibitor was used in a in vitro transcription reaction to to produce capped transcripts. The template DNA was then removed by RQ1 RNase-free DNase. (0328)

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J. Clin. Invest. 104, 647-656. Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice. 1999

Sinz, E.H., Kochanek, P.M., Dixon, C.E., Clark, R.S.B., Carcillo, J.A., Schiding, J.K., Chen, M., Wisniewski, S.R., Carlos, T.M., Williams, D., DeKosky, S.T., Watkins, S.C., Marion, D.W., Billiar, T.R.

Notes: Genomic DNA was isolated from mouse brain with the Wizard® Genomic DNA Purification Kit. The isolated DNA was used for PCR. The RNasin® Ribonuclease Inhibitor was used to protect RNA in RT-PCR. (0372)

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Mol. Cell 1, 765-771. Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing. 1998

Graveley, B.R., Maniatis, T.

Notes: The RNasin® Ribonuclease Inhibitor was used to protect 32P-labeled RNA probes during RNA gel shift assays. (1116)

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J. Biol. Chem. 273, 23454-23462. Identification and characterization of a novel tissue-specific transcriptional activating element in the 5'-flanking region of the CYP2A3 gene predominanatly expressed in rat olfactory mucosa 1998

Zhang, J., Ding, X.

Notes: The Core Footprinting System was used to footprint a region of the CYP2A3 gene with nuclear extracts derived from various rat and mouse tissues. The RNasin® Ribonuclease Inhibitor was used to protect transcripts of a eukaryotic in vitro transcription reaction. The transcripts were used in an S1 Nuclease protection assay. (0091)

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J. Biol. Chem. 273, 10658-10664. Identification of a hormonally regulated luteinizing hormone/human chorionic gonadotropin receptor mRNA binding protein: Increased mRNA binding during receptor down-regulation. 1998

Kash, J.C., Menon, K.M.J.

Notes: The RNasin® Ribonuclease Inhibitor was used to protect transcripts from an in vitro transcription reaction and the same transcripts during RNA gel shift assays. (0917)

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Am. J. Respir. Cell Mol. Biol. 18, 168-178. KGF increases SP-A and SP-D mRNA levels and secretion in cultured rat alveolar type II cells. 1998

Xu, X. , McCormick Shannon, K. , Voelker, D. R. , Mason, R. J.

Notes: The authors use recombinant KGF and RNasin® Ribonuclease Inhibitor in their studies on Alveolar type II cells from Sprague-Dawley rats. (0127)

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Am. J. Hum. Genet. 62, 269-277. Nearby stop codons in exons of the neurofibromatosis type 1 gene are disparate splice effectors. 1998

Hoffmeyer, S., Nurnberg, P., Ritter, H., Fahsold, R., Leistner, W., Kaufmann, D., Krone, W.

Notes: cDNA synthesis was performed in a reverse transcription reaction containing single-stranded binding protein and RNasin® Ribonuclease Inhibitor. In a protein truncation test (PTT), five overlapping segments amplified by RT-PCR of NF1 transcript were expressed by TNT® Coupled Reticulocyte Lysate System. (1051)

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Biochemistry 37(15), 5173-5183. Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties. 1998

Newton, D.L., Boque, L., Wlodawer, A., Huang, C.Y. and Rybak, S.M.

Notes: Naturally isolated onconase, a cellular RNase with anti-tumor properties, is not inhibited by the placental ribonuclease inhibitor. The natural protein has a pyroglutamate at the N-terminus as the result of a posttranslational modification starting from an N-terminal methionine followed by a glutamate residue. Recombinant onconase has no activity unless the N-terminal methionine and the glutamate residue is cyclized to form the pyroglutamate moiety. However, conversion of the glutamate to either serine or tyrosine results in RNase activity without processing. This RNase activity is very different from the natural onconase because the Ser and Tyr mutants are inhibited by RNasin® Ribonuclease Inhibitor. Further studies determined that the Tyr and Ser mutants display a 100,000-fold greater sensitivity to the inhibitor that the natural onconase. (1646)

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J. Immunol. 161, 474-480. System administration of endotoxin induces bronchopulmonary hyperactivity dissociated from TNF-alpha formation and neutrophil sequestration into the murine lungs. 1998

Lefort, J., Singer, M., Leduc, D., Renesto, P., Nahori, M.A., Huerre, M., Créminon, Chignard, M., Vargaftig, B.B.

Notes: Poly A+ RNA was isolated directly from PBS-perfused mouse lungs with the PolyATtract® System 1000. The isolate poly A+ RNA was used for quantitative two-step RT-PCR using M-MLV RNase Hminus Reverse Transcriptase as well as RNasin® Ribonuclease inhibitor for first step cDNA synthesis. (0821)

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Genetics 150, 643-650. Tetrahymena mutants with short telomeres. 1998

Ahmed, S., Sheng, H., Niu, L. and Henderson, E.

Notes: PCR products were excised, eluted, and precipitated prior to sequencing with the fmol® DNA Cycle Sequencing System. Promega's RNasin® Ribonuclease Inhibitor and AMV Reverse Transcriptase were used in a reverse transcription assay. (2065)

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J. Biol. Chem. 273, 5607-5614. Transcriptional activation of the UDP-glucuronosyltransferase 1A7 gene in rat liver by aryl hydrocarbon receptor ligands and oltipraz. 1998

Metz, R.P. and Ritter, J.K

Notes: The RNasin Ribonuclease Inhibitor was used to protect RNA transcripts during primer extension analysis and in vitro transcription of RNA probes. The resultant RNA probes were used for RNase protection assays. (1639)

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