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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. 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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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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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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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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S1 Nuclease

Biochem. Biophys. Res. Commun. 408, 160-166. Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells. 2011

 Yu, K.S., Jo, J.Y., Kim, S.J., Lee, Y., Bae, J.H., Chung, Y.H., and Koh, S.S.

Notes: These authors showed that expression of the serine protease inhibitor elafin is regulated by epigenetically controlled expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, resulting in reduced proliferation and increased apoptosis. Luciferase reporter assays were used to show that Foxa2 binding was required for activation of elafin expression, and that Foxa2 binding was activated by treatment with the methyltransferase inhibitor. These assays used a pGL3-Basic Vector construct in which expression of firefly luciferase was driven by the upstream region bearing the Foxa2 binding site. The pRL-TK Vector, expressing Renilla luciferase, was used as a normalization control. The AccessQuick™ System was used for RT-PCR analysis to show that Foxa2 mRNA was barely detectable in melanoma cells.  (4345)

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PLos ONE 6(9), e24417. Metagenome plasticity of the bovine abomasal microbiota in immune animals in response to Ostertagia ostertagi infection. 2011

Li, R.W., Wu, S., Li, W., Huang, Y., and Gasbarre, L.C.

Notes: These authors performed metagenomic analysis to investigate any changes in composition of the microbial flora of the abomasa (ruminant 4th stomach compartment) in immune cattle after reinfection with the nematode Ostertagia ostertagi. DNA was extracted from abomasal contents using a QIAamp stool kit and the integrity verified using an Agilent Bioanalyzer 2000. PCR was then performed using primers targeting hypervariable regions of 16S rRNA genes. The amplified material was gel-purified and sequenced using the Roche 454 pyrosequencing system. DNA concentration was measured before and after PCR using the QuantiFluor™ Fluorometer.


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Bioinformatics 27, 2027-2030. Pathogen detection using short-RNA deep sequencing subtraction and assembly 2011

Isakov, O., Modai, S. and Shomron, N.

Notes: To confirm the  Mycoplasma contamination in HIV-1 infected samples detected by high-throughput sequencing, a PCR-based Mycoplasma test was performed. Products were separated on an agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System before confirmatory sequencing. (4537)

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PLos ONE 6, e29604. Probing the SELEX process with next-generation sequencing 2011

Schütze, T., Wilhelm, B., Greiner, N., Braun, H., Franziska, P., Möri, M., Erdman, V.A., Lehrach, H., Konthur, Z., Menger, M., Arndt, P.F. and Glökler, J.

Notes: Wizard® SV Gel and PCR Clean-Up System was used to purify products after barcodes were attached by PCR prior to sequencing on an Illumina Genome Analyzer GA2. (4553)

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Infect. Immun. 79, 2451-9. The Agr-like quorum-sensing system regulates sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A non-food-borne human gastrointestinal disease strain F5603. 2011

Li, J., Chen, J., Vidal, J.E., and  McClane, B.A.

Notes: This study explored whether the Agr-like quorum-sensing system regulates sporulation and production of the Clostridium perfringens toxins CPE and beta2 toxin. An agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, had reduced production of alpha-toxin and perfringolysin O during vegetative growth, and did not form spores efficiently. The AccessQuick™ System was used in RT-PCR analysis to confirm the presence or absence of agrB transcripts in the wild type, mutant, and complemented strains used in the study. (4546)

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DNA Research 18, 93–105. The complete chloroplast genome of 17 individuals of pest species Jacobaea vulgaris: SNPs, microsatellites and barcoding markers for population and phylogenetic studies 2011

Doorduin, L., Gravendeel, B., Lammers, Y., Ariyurek, Y., Chin-A-Woeng, T. and Vrieling, K.

Notes: Wizard® SV Gel and PCR Clean-Up System was used to extract and purify DNA fragments that had been amplified by long-range PCR and separated on an agarose gel prior to next-generation sequencing on an Illumina platform. (4535)

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

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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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 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.

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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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J. Microbiol. Methods 81, 127-134. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. 2011

Salonen, A., Nikkilä, J., Jalanka-Tuovinen, J., Immonen, O., Rajilić-Stojanović, M., Kekkonen, R.A., Palva, A., and de Vos, W.M.

Notes: These authors compared the performance of four DNA purification methods for recovery of bacterial and archaeal DNA from fecal material. One of the methods tested was the Wizard® Genomic DNA Purification Kit, which uses a solution-based, enzymatic method for extraction. The Wizard® Genomic method was rated highly on extraction speed, and gave the highest DNA yields. A second method involving mechanical disruption (repeated bead beating) was rated more highly on extraction efficiency from archaea and some bacterial species. The criteria for performance comparison are described fully in the paper. (4219)

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Appl. Environ. Microbiol. 77, 2589–95. Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification. 2011

Han, F., Wang, F. and Ge, B.

Notes: The authors developed a loop-mediated isothermal amplification (LAMP) assay to distinguish between virulent and nonvirulent strains of Vibrio vulnificus by targeting the virulence-correlated gene (vcg). The authors performed PCR using vcg-specific primers and GoTaq® Hot Start Polymerase in parallel to confirm the LAMP results. (4163)

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J. Clin. Microbiol. 49(1), 335-44. Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.

Medina, R.A., et al.

Notes: These authors used extensive computational analysis of isolates from the 2009 H1N1 outbreak to identify conserved H1N1 sequence signatures that could potentially be used in diagnostic assays to track the spread of specific strains during viral outbreaks. They identified target sequences in the hemagglutinin and neuraminidase genes that were highly conserved among 2009 H1N1 isolates. They then designed primers to amplify those sequences and used them in Taqman® and SYBR® Green-based qPCR assays to create a discriminatory 2009 H1N1 detection assay. They used the AccessQuick™ System in conventional RT-PCR to first establish whether their chosen primers were specific for the 2009 H1N1 isolates. In that assay they amplified representative H1N1 strains spanning from 1930 to the 2009 H1N1, and showed that only the 2009 isolates generated product. (4284)

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Proc. Natl. Acad. Sci. USA 108, 18488–93. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. 2011

Allen, J.A., Yost, J.M., Setola, V., Chen, X., Sassano, M.F., Chen, M., Peterson, S., Yadav, P.N., Huang, X.P., Feng, B., Jensen, N.H., Che, X., Bai, X., Frye, S.V., Wetsel, W.C., Caron, M.G., Javitch, J.A., Roth, B.L. and Jin, J.

Notes: This paper explored potential compounds as agonists of dopamine D2 receptor (D2R) with a bias toward β-arrestin signaling. Based on the aripiprazole scaffold, compounds were synthesized and tested in a D2-mediated Gi-coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R transfected with pGloSensor™-22F cAMP Plasmid. Assessing β-arrestin recruitment to agonist-stimulated receptors was determined using HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase exposed to agonist or D2 test ligand with or without reference agonist. After 18 hours, medium was removed from the cells, 1X Bright-Glo™ Reagent added and luminescence measured. (4518)

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