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J. Biotechnol. 157(4), 598-604. Detection of toxic lignin hydrolysate-related compounds using an inaA::luxCDABE fusion strain. 2012

Lee, S., and Mitchell, R.J.

Notes: This study evaluated use of a luciferase reporter construct for detection of hydrolysate related phenolic compounds generated during fermentation of plant products. The authors used a transcriptional fusion of the inaA promoter with the luxCDABE operon to detect phenolic compounds generated in plant hydrolysates. The GloMax® Multi Detection System was used to measure bioluminescence in various bacterial cultures. (4194)

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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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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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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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Antimicrob. Agents Chemother. 56, 2504–10. Expression of the resistance-nodulation-cell division pump AdelJK in Acinetobacter baumannii is regulated by AdeN, a TetR-type regulator. 2012

Rosenfeld, N. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Acinetobacter baumannii. (4296)

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Appl. Environ. Microbiol. 78, 5717–5723. High-throughput sequencing for detection of subpopulations of bacteria not previously associated with artisanal cheeses 2012

Quigley, L., O'Sullivan, O., Beresford, T., Ross, R.P., Fitzgerald, G.F. and Cotter, P.D.

Notes: GoTaq® Green Master Mix was used in the PCR amplification of genomic DNA template for pyrosequencing. (4541)

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Antimicrob. Agents Chemother. 56, 5332–9. Identification of a novel genomic island conferring resistance to multiple aminoglycoside antibiotics in Campylobacter coli. 2012

Qin, S. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Campylobacter coli. (4316)

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Gene 507, 152–8. Identification of novel transcripts deregulated in buccal cancer by RNA-seq. 2012

Sajnani, M.R., Patel, A.K., Bhatt, V.D., Tripathi, A.K., Ahir, V.B., Shankar, V., Shah, S., Shah, T.M., Koringa, P.G., Jakhesara, S.J. and Joshi, C.G.

Notes: The authors used the Roche 454 sequencing platform to perform a differential transcriptome analysis and identify genes that are differentially expressed in human buccal cancer and normal tissue. The quantity of first-strand and second-strand cDNA products was estimated using the QuantiFluor™-ST Fluorometer as well as the High Sensitivity DNA Chip kit and Agilent Bioanalyzer 2100. (4238)

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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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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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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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