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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 transcription 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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DNA Research 19, 395-406. Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer 2012

Murphy, S.J., Cheville, J.C., Zarei, S., Johnson, S.H., Sikkink, R.A., Kosari, F., Feldman, A.L., Eckloff, B.W., Karnes, R.J. and Vasmatzis, G.

Notes: Genomic rearrangements detected by NGS were mapped and confirmed by PCR. Human Genomic DNA: Male was used as a control for the confirmatory experiments. (4539)

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Biochim. Biophys. Acta 1822, 248-260. MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20. 2012

Yao, T., and Lin, Z.

Notes: This study identified that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines, and showed that the level of miR-21 correlates with tumor differentiation, and regulates proliferation, apoptosis, and migration of HPV16-positive cells. The authors used gene expression profiling and luciferase reporter assay to identify candidate target genes for miR-21. Wildtype and mutant 3′-UTR sequences from the target gene CCL20 containing putative binding sites for miR-21 were subcloned into the psiCHECK-2 Vector and used to transfect HEK293 cells. The Dual-Luciferase® Reporter Assay System and GloMax® Multi Luminometer were used to measure luciferase activity from both constructs. (4192)

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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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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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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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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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New Phytol. 195, 844–56. Seasonal trends in the biomass and structure of the bryophyte-associated fungal communities explored by 454 pyrosequencing 2012

Davey, M.L., Heegaard, E., Halvorsen, R., Ohlson, M. and Kauserud, H.

Notes: Genomic DNA was extracted from shoot fragments using an organic extraction procedure and purified using the Wizard® SV Gel and PCR Clean-Up System prior to template preparation by nested PCR. Products from the second PCR were separated by electrophoresis and purified using the Wizard® SV Gel and PCR Clean-Up System prior to pyrosequencing. (4548)

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J. Clin. Microbiol. 50(5), 1580–5. Sensitive and rapid detection of the New Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification. 2012

Liu, W. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Acinetobacter baumannii. (4274)

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J. Biomed. Sci. 19, 42. Shikonin enhances efficacy of a gene-based cancer vaccine via induction of RANTES. 2012

Chen, H.M., Wang, P.H., Aravindaram, K., Chen, Y.H., Yu, H.H., Yang, W.C., and Yang, N.S.

Notes: In this study, the authors evaluated whether application of the phytochemical shikonin to the skin of mice was able to augment the effect of a DNA-based anti-tumor vaccine by inducing the cytokine RANTES. As part of the study, the AccessQuick™ System was used in RT-PCR analysis to determine expression of RANTES mRNA in treated and control skin samples. (4288)

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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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J. Virol. 86, 10999–11012. Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces. 2012

Wu, Z., Ren, X., Yang, L., Hu, Y., Yang, J., He, G., Zhang, J., Dong, J., Sun, L., Du, J., Liu, L., Xue, Y., Wang, J., Yang, F., Zhang, S. and Jin, Q.

Notes: Swab samples from 11 species of Chinese bats were vortexed in maintenance medium, filtered through a 0.45µm pore filter, centrifuged and resuspended. Any nonencapsidated (naked) nucleic acid was digested in a cocktail of enzymes including 20U of RNase ONE™ Ribonuclease prior to DNA and RNA purification. Conserved regions of the RNA-dependent RNA polymerase gene of astroviruses were reverse transcribed, amplified and cloned into the pGEM®-T Easy Vector for sequencing. (4552)

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J. Biol. Chem. 286, 37196–206. 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-myc nuclear localization and binding to the e-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes. 2011

Ning, B., Liu., G., Liu, Y., Su, X., Anderson, G.J., Zheng, X., Chang, Y., Guo, M., Liu, Y., Zhao, Y. and Nie, G.

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

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Nucl. Acids Res. 39, e81. A method for counting PCR template molecules with application to next-generation sequencing.
2011


J.A. Casbon, R. J. Osborne, S. Brenner and C.P. Lichtenstein

Notes: Human Genomic DNA used as the starting material in the NGS workflow.  GoTaq® Flexi Colorless Buffer and GoTaq® Flexi Polymerase were used in amplification of template in step added to the beginning of library preparation. (4531)

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Nucl. Acids Res. 39, e81. A method for counting PCR template molecules with application to next-generation sequencing. 2011

Casbon, J.A., Osborne, R.J., Brenner, S. and Lichtenstein, C.P.

Notes: DNA templates are often amplified by PCR during library generation prior to next-generation sequencing, but amplification can introduce biases and duplications that are not easily corrected. In this paper, the authors developed a simple method to count the number of input template molecules to reduce these PCR-related problems: The ligation of a degenerate base region to all fragments during library creation. To evaluate their approach to correct for biases and duplications, the authors created a library using Human Genomic DNA, amplified the library by inverse PCR using the GoTaq® Hot Start Polymerase and 1X Colorless GoTaq® Flexi Buffer, sequenced the resulting DNA fragments and assessed the quality of the next-generation sequencing data. (4160)

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Hum. Mol. Genet. 21, 577–85. A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing 2011

Soldà, G., Robusto, M., Primignani, P., Castorina, P., Benzoni, E., Cesarani, A., Ambrosetti, U., Asselta, R. and Duga, S.

Notes: To confirm the role of a mutation in the miR-96 microRNA (miRNA) associated with an autosomal dominant hearing lost, HeLa cells (250,000 cells per well in six-well plates) were transfected with 4µg of plasmid carrying wild type or mutant miR-96 miRNA using FuGENE® HD Transfection Reagent. After 24 hours, the cells were washed and total RNA extracted. After quantitation, the RNA used in RT-PCR analysis. The entire 3´UTRs of eight putative target genes were amplified by PCR from genomic DNA and cloned into the psiCHECK™-2 Vector. HeLa cells were transiently transfected with 2µg of the 3´ UTR psiCHECK™-2 constructs and 0.2µg of a wild-type, single or double mutant miR-96 plasmid using FuGENE® HD Transfection Reagent. Forty-eight hours after transfection, the Dual-Luciferase® Reporter Assay System was used to quantify the firefly and Renilla luciferase in cell lysates. (4251)

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J. Biol. Chem. 286, 42690-42703. Alternative Splicing Produces Nanog Protein Variants with Different Capacities for Self-renewal and Pluripotency in Embryonic Stem Cells. 2011

Das, S., Jena, S., and Levasseur, D.N.

Notes: The transcription factor Nanog is required for the maintenance of embryonic stem (ES) cell pluripotency. These authors showed that the Nanog N-terminal domain is regulated by post-transcriptional modification, and that alternative splicing generates Nanog variants with different capacities for maintaining an undifferentiated cell state. As part of their study, the authors used GoScript® Reverse Transcriptase to generate cDNA from RNA extracted from cell lines expressing different Nanog variants. The cDNA was used in RT-qPCR to quantify relative expression levels. (4184)

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PLos ONE 6, e25263. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform 2011

Tamaki, H., Wright, C.L., Li, X., Lin, Q., Hwang, C., Wang, S., Timmapuram, J., Kamagata, Y. and Liu, W.T.

Notes: DNA was isolated from a variety of environmental samples including surface soil, drinking water biofilm, sludge from an anaerobic digester, bioreactor samples, ground water, peat soil and glacial deposit soil. The 16S rRNA gene was amplified from the DNA. PCR amplifications were run on agarose gels, and bands of the predicted sizes excised and purified using the Wizard® SV Gel and PCR Clean-Up System before pooling for pyrosequencing. (4554)

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J. Clin. Microbiol. 49, 281–291. Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers. 2011

Stressmann, F.A., Rogers, G.B., Klem, E.R., Lilley, A.K., Donaldson, S.H., Daniels, T.W., Carroll, M.P., Patel, N., Forbes, B., Boucher, R.C., Wolfgang, M.C. and Bruce, K.D.

Notes: Sputum samples were collected from cystic fibrosis patients and 16S rRNA sequences amplified by PCR. These products were cloned into a T-vector, transformed into competent cells and the resulting colonies grown in 2ml LB broth in 96-deep-well plate for 20 hours. Of this culture, 1.9ml was pelleted and the clones isolated using the Wizard® SV Plasmid Purification System. The purified plasmid DNA was subjected to agarose gel electrophoresis and sequenced. (4133)

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Genome Res. 21, 1738-45. Application of microdroplet PCR for large-scale targeted bisulfite sequencing 2011

Komori, H.K., LaMere, S.A., Torkamani, A., Hart, G.T., Kotsopoulos, S., Warner, J., Samuels, M.L., Olson, J., Head, S.R., Ordoukhanian, P., Lee, P.L., Link, D.R. and Salomon, D.R.

Notes: The authors of this study sought to correlate promoter methylation with gene expression. Gene expression data was generated by RNA-seq of Jurkat cells. Amplified cDNA was prepared from total RNA, the cDNA was treated with S1 nuclease to remove single stranded nucleic acids and used as template for the sequencing libraries. (4536)

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Blood Cells Mol. Dis. 46, 139-144. Application of MLPA assay to characterize unsolved α-globin gene rearrangements. 2011

Colosimo, A,. Gatta, V., Guida, V., Leodori, E., Foglietta, E., Rinaldi, S., Cappabianca, M.P., Amato, A., Stuppia, L., and Dallapiccola, B.

Notes: These authors used the Maxwell® 16 Blood DNA Purification Kit to isolate genomic DNA from leukocytes. (4210)

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Acta Pharmacologica Sinica 32, 368-74. Attenuated Salmonella typhimurium carrying shRNA-expressing vectors elicit RNA interference in murine bladder tumors. 2011

Yang, N., Li, S.H., Lü, Y.Z., Chen, L.S., and Ren, D.M.

Notes: This proof-of-principle study investigated whether attenuated Salmonella typhimurium could be used as a vehicle for delivering shRNA-expressing plasmid DNA into cancer cells in mice. The authors delivered S. typhimurium bearing plasmids encoding anti-GFP shRNA orally to mice harboring tumors that expressed GFP. They were able to show that the bacteria accumulated and persisted for 40 days within the tumors, and that GFP expression in infected tumors was reduced. The AccessQuick™ RT-PCR System was used to analyze GFP expression levels in cultured cells and tumors. (4347)

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