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FASEB J. 28, 946–55. Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements. 2014

Zulueta, A., Caretti, A., Signorelli, P., Dall'olio, F. and Trinchera, M.

Notes: RNA was isolated from several cell lines using the SV Total RNA Isolation System and ReliaPrep™ RNA Cell Miniprep System. RNA was used in competitive RT-PCR to characterize splice variants of the B3GALT5 long terminal repeat, HNFα and HNFβ1. (4445)

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Epigenetics 8, 534–541. First evidence of DNA methylation in insect Tribolium castaneum: Environmental regulation of DNA methylation with heterochromatin 2013

Feliciello, I., Parazajder, J., Akrap, I. and Ugarović, Ð.

Notes: GoTaq® Green Master Mix was used to amplify Tribolium castaneum  satellite DNA that had been bisulfite treated to detect methylated cytosines. The bisulfite-treated satellite DNA was amplified using methyl-specific primers in a total volume of 30µl using 2X GoTaq® Green Master Mix, 2mM mix of the primer sets, and 1µl of the bisulfite modified DNA.

  (4356)

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Cell 154, 541–555. KDM4A Lysine Demethylase Induces Site-Specific Copy Gain and Rereplication of Regions Amplified in Tumors 2013

Black, J.C., Manning, A.L., Van Rechem, C., Kim, J., Ladd, B., Cho, J., Pineda, C.M., Murphy, N., Daniels, D.L., Montagna, C., Lewis, P.W., Glass, K., Allis, C.D., Dyson, N.J., Getz, G. and Whetstine, J.R.

Notes: The HaloTag® protein tag was used in experiments to identify protein partners of KDM4A that involved in site-specific copy number gain in tumors, specifically at the 1q12h region. Expression constructs were transfected into HEK293T cells using the FuGENE® HD Transfection Reagent. The HaloTag-KDM4A (Cat.# FHC00602) and HaloTag-Suv39h1 (Cat.# FHC09879) expression constructs were obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). Interacting proteins identified included DNA polymerase subunits and members of the minichromosome maintenance (MCM) complex. (4408)

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Proc. Natl. Acad. Sci. USA 110, 18620–5. GATA-1 regulates the generation and function of basophils. 2013

Nei, Y., Obata-Ninomiya, K., Tsutsui, H., Ishiwata, K., Miyasaka, M., Matsumoto, K., Nakae, S., Kanuka, H., Inase, N. and Karasuyama, H.

Notes: RNA was isolated from mouse basophils following fluorescence-activated cell sorting and used in RT-qPCR analysis. (4443)

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Nucl. Acids Res. 41, 8515–25. Pin1 promotes GR transactivation by enhancing recruitment to target genes. 2013

Poolman, T.M., Farrow, S.N., Matthews, L., Loudon, A.S. and Ray, D.W.

Notes: RNA was extracted from cultured A549 cells and used in RT-qPCR to analyze the effect of transfected siRNAs. (4444)

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Anal. Biochem. 423(2), 224-228. A bioluminescence assay for DNA methyltransferase activity based on methylation-resistant cleavage. 2012

Jiang, C., Yan, C.Y., Huang, C., Jiang, J.H., and Yu, R.Q.

Notes: This paper describes a bioluminescence-based method for the detection of DNA methyltransferase activity based on methylation-resistant cleavage and protein expression. The authors used Dam methylase as a model enzyme, MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) as the target. LR-DNA was amplified by PCR and then used as substrate in a Dam methylation reaction. If the target sites in the LR-DNA were fully methylated, they were resistant to subsequent MboI cleavage and expressed luciferase upon in vitro transcription/translation. Incomplete methylation or the absence of methylation resulted in DNA digestion and diminished/absent luciferase activity. TNT® T7 Quick for PCR DNA was used for in vitro translation of the LR-DNA, and the Luciferase Assay System and GloMax® 20/20 Luminometer were used to measure luciferase activity. (4191)

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PLos ONE 7, e48183. Enhanced expression of vacuolar H+-ATPase subunit E in the roots is associated with the adaptation of Broussonetia papyrifera to salt stress. 2012

Zhang, M., Fang, Y., Liang, Z. and Huang, L.

Notes: The authors examined cellular adaptation to increased salinity in Broussonetia papyrifera by measuring protein and mRNA levels of vacuolar H+-ATPase (V-H+-ATPase) subunits and the activities of V-H+-ATPase and vacuolar H+-pyrophosphatase. Relative expression levels of V-H+-ATPase subunits A, B, E and c in salt-stressed and control plants were determined by RT-PCR using actin as a normalization control gene. cDNA was synthesized using the GoScript™ Reverse Transcription System as described by the manufacturer’s protocol, then amplified by PCR for 25 cycles. The amplification products were analyzed and quantified by agarose gel electrophoresis and ethidium bromide staining. (4258)

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PLos ONE 7, e47892. Epigenetic disruption of the PIWI pathway in human spermatogenic disorders. 2012

Heyn, H., Ferreira, H.J., Bassas, L., Bonache, S., Sayols, S., Sandoval, J., Esteller, M. and Larriba, S.

Notes: The authors used microarray analysis, bisulfite sequencing and pyrosequencing to examine a possible link between aberrant DNA methylation and abnormal human spermatogenesis and male infertility. They identified almost 600 genes that were differentially methylated in testis tissue of men with secretory male infertility. Genomic DNA used in the microarray analysis was extracted from testicular biopsies using the Wizard® Genomic DNA Purification Kit. For the bisulfite sequencing experiments, genomic DNA was bisulfite-modified, amplified, cloned using the pGEM®-T Easy Vector System, then sequenced. (4257)

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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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Human Pathology in press. Proliferative glomerulonephritis with monoclonal immunoglobulin G3κ deposits in association with parvovirus B19 infection 2012

Fujita, E., Shimizu, A., Kaneko, T., Masuda, Y., Ishihara, C., Mii, A., Higo, S., Kajimoto, Y., Kanzaki, G., Nagasaka, S., Iino, Y., Katayama, Y. and Fukuda, Y.

Notes: The authors used the ReliaPrep™ FFPE gDNA Miniprep System to isolate DNA from parafin-embedded kidney sections for use in real-time PCR to detect parvovirus B19. (4235)

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Cancer Res. 72, 810-820. SMYD3 Promotes Cancer Invasion by Epigenetic Upregulation of the Metalloproteinase MMP-9. 2012

Cock-Rada, A.M., Medjkane, S., Janski, N., Yousfi, N., Perichon, M., Chaussepied, M., Chluba, J., Langsley, G., and Weitzman, J.B.

Notes: These authors investigated the role of matrix metalloproteinase (MMP-9) in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. They found that gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. The H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. They therefore investigated the role of SMYD3 overexpression on MMP-9 expression and cell migration, identifying SMYD3 as an important new regulator of MMP-9 transcription. During the study they used the GloMax® Multi Luminometer to measure luminescence and absorbance in reporter and cell viability assays. They also used the Dual-Luciferase® Reporter Assay to measure SMYD3 activity in cells transfected with a SMYD3 reporter, and the pGL4-hRluc/TK plasmid for normalization of the experimental reporter activity. GoTaq® DNA polymerase was used in semi-quantitative RT-PCR. (4189)

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Vet. Parasitol. 188, 160–3. Use of a real time PCR for detecting subspecies of Babesia canis. 2012

Costa, L.M., Jr. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Babesia canis. (4311)

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mBio. 3(5), e00266–12. A multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus. 2012

Alter, H.J., Mikovits, J.A., Switzer, W.M., Ruscetti, F.W., Lo, S.C., Klimas, N., Komaroff, A.L., Montoya, J.G., Bateman, L., Levine, S., Peterson, D., Levin, B., Hanson, M.R., Genfi, A., Bhat, M., Zheng, H., Wang, R., Li, B., Hung, G.C., Lee, L.L., Sameroff, S., Heneine, W., Coffin, J., Hornig, M. and Lipkin, W.I.

Notes: In this report, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with Chronic Fatigue Syndrome (CFS) report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects.

The CDC performed nucleic acid testing assays. Plasma was centrifuged and RNA isolated from the pellet. Quantitative real-time RT-PCR assays (qRT-PCR) for generic pMLV/XMRV pro (protease) and gag detection were performed on RNA extracts, using the AccessQuick™ RT-PCR System and an AgPath one-step RT-PCR kit.

ArrayScript RT and AmpliTaq Gold DNA polymerase were used for cDNA synthesis and amplification in the pro and gag qRT-PCR assays, respectively. A third PCR was done using the primers XPOLOF and XPOLOR, followed by a nested PCR with the primers XPOLIF and XPOLIR for the generic detection of MLV/XMRV 216-bp pol sequences. For this reaction, cDNA synthesis and amplification of RNA was done using Promega AMV Reverse Transcriptase and a RobustI RT-PCR kit. Each PCR experiment included 20 water-only reactions to control for contamination. (4300)

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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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Viruses 4, 200–10. Clinical characteristics and genetic variability of human rhinovirus in Mexico. 2012

Landa-Cardeña, A., Morales-Romero, J., García-Roman, R., Cobián-Güemes, A.G., Méndez, E., Ortiz-Leon, C., Pitalúa-Cortés, F., Mora, S.I. and Montero, H.

Notes: This study examined the prevalence of strains of human rhinovirus (HRV) that may be causing respiratory infections in Mexican children. Nucleic acids were purified from nasal swabs of two-year-old children, and screened for the presence of HRV by amplifying 20ng of HRV-RNA using the AccessQuick™ RT-PCR System with primers for the 5´ nontranslated region. Products were sequenced and aligned with sequences found in GenBank. (4343)

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Food Control 23, 400-404. Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures. 2012

Cammà, C., Di Domenico, M., and Monaco, F.

Notes: These authors used the Maxwell® 16 Tissue DNA Purification Kit to extract DNA from 200µl samples of raw meat homogenate from beef, pork, poultry and lamb samples. They also used the Wizard® Genomic DNA Isolation System to extract DNA from cooked meat samples. The extracted DNA was used in real-time, quantitative PCR assays to identify species-specific DNA spiked at 1% in mixed DNA samples. (4353)

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Am.J.Trop. Med. Hyg. 86(4), 732–735. Identification of Oropouche Orthobunyavirus in the Cerebrospinal Fluid of Three Patients in the Amazonas, Brazil. 2012

Bastos, M.de S., Figueiredo, L.T., Naveca, F.G., Monte, R.L., Lessa, N., Pinto de Figueiredo, R.M., Gimaque, J.B., Pivoto João, G., Ramasawmy, R. and Mourão, M.P.

Notes: Oropouche fever is a arboviral infection in Brazil, surpassed in frequency only by dengue. Oropouche virus (OROV) causes large outbreaks of acute febrile illness in areas along the Amazon and Central-Plateau regions. RNA was extracted from CSF and underwent reverse transcription-polymerase chain reaction and sequencing to identify OROV. Reverse transciption was performed with 5ml of the random primers, using the AccessQuick™ RT-PCR System. (4320)

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Virol. J. 9, 144. Inaccurate identification of rotavirus genotype G9 as genotype G3 strains due to primer mismatch. 2012

Mitui, M.T., Chandrasena, T.N., Chan, P.K., Rajindrajith, S., Nelson, E.A., Leung, T.F., Nishizono, A. and Ahmed, K.

Notes: This study examined how well primers developed in 1990 and 2004 for type A rotavirus (RVA) were able to genotype (G type) currently circulating RVAs in Asia. The VP7 gene from RVA was amplified using 2µl of dsRNA template with the AccessQuick™ RT-PCR System in a total volume of 50µl. The G type was determined using hemi-nested multiplex PCR using 1µl of the VP7 cDNA and PCR Master Mix in a final volume of 50µl. The final products were sequenced. (4340)

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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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Int. J. Infect. Dis. 16(1), e53–9. Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India. 2012

Shukla, J., Saxena, D., Rathinam, S., Lalitha, P., Joseph, C.R., Sharma, S., Soni, M., Rao, P.V. and Parida, M.

Notes: This study describes the clinical observations and laboratory investigations performed on 170 of the 2,000 suspected West Niles Virus (WNV) cases. These cases were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription PCR (RT-PCR) and real-time RT-PCR assays were used to detect WNV infection. In addition, reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) was performed to determine the feasibility of using this method as an alternative cost-effective tool to the real-time RT-PCR.

After proving negative for DENV- and CHIKV, samples were tested for the presence of WNV-specific RNA by RT-PCR, real-time RT-PCR and RT-LAMP assays. RNA was extracted from the patient serum, plasma and infected culture supernatant. The RNA was then eluted in 50µl of nuclease-free water and used as template in the AccessQuick™ RT-PCR System, with primer pairs targeting the env gene.

Amplification was performed in a 50µl total reaction volume with the AccessQuick™ RT-PCR System, using 50pmol of each forward and reverse primer and 2µl of extracted viral RNA. (4331)

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Veterinary Microbiology 160(3-4), 463–467. Molecular detection of murine noroviruses in laboratory and wild mice. 2012

Farkas, T., Fey, B., Keller, G., Martella, V. and Egyed, L.

Notes: Mice RNA samples were converted to cDNA using an oligo-dT primer with the Reverse Transcription System, ethanol precipitated, vacuum dried and transferred to another lab. There they were reconstituted in 20μl of molecular biology grade water.

Detection of caliciviruses in the wild mice samples was attempted using generic calicivirus primers targeting sequences encoding conserved amino acid motifs in the RNA-dependent RNA polymerase (RdRp) region of ORF1. Two microliters of cDNA was used in 25μl PCR reactions using the GoTaq® Green Master Mix. Laboratory mouse RNA samples were tested only with MNV-specific primers in the AccessQuick™ RT-PCR System using 2μl RNA as template.

PCR products were cloned into pGEM-T® Vector and sequenced using M13 forward and reverse primers on an ABI PRISM® 3730 DNA Analyzer. (4330)

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PLoS Genet. 8, e1002941. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells. 2012

Goodier, J.L., Cheung, L.E. and Kazazian, H.H. Jr.

Notes: To better understand the role MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), in reducing retrotransposon activity, 293T human embryonic kidney cells expressing MOV10 and one of three retrotransposons (LINE1 (L1), Alu or SVA) were lysed in a buffer that included RNasin® Ribonuclease Inhibitor, and FLAG-tagged L1 complexes immunoprecipitated and analyzed by mass spectrometry. Several retrotransposon assays were conducted using FuGENE® HD Transfection Reagent for transfected constructs into 293T, HeLa and HeLa-HA cells. (4255)

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J. Immunol. 188(4), 1896–1904. Plac8-dependent and inducible NO synthase-dependent mechanisms clear Chlamydia muridarum infections from the genital tract. 2012

Johnson, R.M., Kerr, M.S. and Slaven, J.E.

Notes: The authors previously showed that there are two independent mechanisms by which Chlamydia-specific CD4 T cells clear infection in epithelial cells; an iNOS-dependent mechanism and a Plac8-dependent mechanism. To further identify the Plac8 mechanism, they used microarrays to identify a second mechanism dependent on Plac8 for terminating Chlamydia replication in epithelial cells.

Several Chlamydia-specific CD4 T cell clones were purified at the end of their culture cycle and grown for 3 days in their usual culture media plus growth factors, without Ag stimulation. Total RNA was isolated from each clone using a protocol that included an RNase-free DNase I treatment step. Specific mRNA gene reverse transcription and amplification were performed using the AccessQuick™ RT-PCR System. (4324)

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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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J. Biomol. Tech. 23, 4-10. Random amplification and pyrosequencing for identification of novel viral genome sequences. 2012

Hang, J., Forsheym, B.M., Kochel, T.J., Li, T., Solórzano, V.F., Halsey, E.S., and Kuschner, R.A.

Notes: This paper describes a method for sequencing unknown viral isolates from tissue culture using anchored random reverse transcription and PCR, pyrosequencing and data analysis. RNA was extracted from tissue culture supernatants positive for viral antigens and used in RT-PCR with random primers. Amplification products were gel-purified and used in pyrosequencing reactions. A QuantiFluor™-P Fluorometer was used to measure copy number concentration relative to a standard, prior to Roche 454 pyrosequencing. (4231)

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