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J. Immunol. 178, 986-92. Identification of CXCL11 as a STAT3-dependent gene induced by IFN. 2007

Yang, C.H., Wei, L., Pfeffer, S.R., Du, Z., Murti, A., Valentine, W.J., Zheng, Y. and Pfeffer, L.M.

Notes: The STAT proteins are involved in the transcriptional response to interferon (IFN), which includes induction of CXCL11 and ISG15 genes. The authors used quantitative real-time PCR to examine CXCL11 and ISG15 expression levels in IFN-sensitive and IFN-resistant cells after IFN treatment. Expression levels of the IFN-responsive genes were normalized to that of β-actin. Total RNA was isolated from untreated and IFN-treated cells, and qPCR was performed on a Bio-Rad iCycler® instrument using the AccessQuick™ RT-PCR System and SYBR® Green I. Reverse transcription was performed at 48°C for 45 minutes, followed by 35 cycles of PCR. Prior to qRT-PCR, PCR product size was confirmed by agarose gel electrophoresis, and PCR specificity was checked by analyzing melting curves. (3767)

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Microbiology 153, 3023–3033. Expression analysis of extracellular proteins from Phanerochaete chrysosporium grown on different liquid and solid substrates. 2007

Sato, S., Liu, F., Koc, H. and Tien, M.

Notes: The authors characterized expression of extracellular proteins by white-rot fungus, Phanerochaete chrysosporium, grown on wood. Temporal expression of these proteins was monitored by relative quantitative RT-PCR. Two micrograms of total RNA was reversed transcribed using 1µg of Random Primers at 37°C for 1 hour. PCRs with one set of PCR primers were performed using 0.5 units of GoTaq® DNA Polymerase, 1X reaction buffer, 250µM each dNTP, 0.5µM each primer and 1µl of cDNA. PCRs with two sets of PCR primers were performed using 2.5 units of GoTaq® DNA Polymerase, 1.6X reaction buffer, 500µM each dNTP, 0.5µM each primer and 1µl of cDNA. (3708)

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Mol. Biol. Cell 18, 2795–804. A developmentally regulated chaperone complex for the endoplasmic reticulum of male haploid germ cells. 2007

van Lith, M., Karala, A.R., Bown, D., Gatehouse, J.A., Ruddock, L.W., Saunders, P.T. and Benham, A.M.

Notes: PDILT is a protein disulfide isomerase (PDI) homolog under developmental control and is induced during puberty. To determine whether PDILT expression coincides with the first wave of spermatogenesis, the authors examined expression of PDILT in 2-, 15-, 30-, 45- and 58-day old rats using RT-PCR. Total RNA was isolated from rat testis, and 50ng was amplified using the AccessQuick™ RT-PCR System and primer pairs specific for PDILT, PDI and calmegin, a testis-specific chaperone that is expressed upon the appearance of spermatocytes. Primer pairs were designed to span introns to avoid amplification of genomic DNA. PCR products were analyzed on a 1% agarose gel. Results showed that PDILT mRNA was first detected during the onset of spermatogenesis. (3765)

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Can. J. Vet. Res. 71, 230–235. Presence of non-O157 Shiga toxin-producing Escherichia coli in feces from feedlot cattle in Alberta and absence on corresponding beef carcasses. 2007

Renter, D.G., Bohaychuk, V., Van Donkersgoed, J. and King, R.

Notes: Beef carcasses were tested for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC) to determine prevalence and serotypes. Carcasses were swabbed with a sponge, incubated in brain–heart infusion broth and 300µl of growth transferred to a separate tube. The bacteria were diluted in water, spun and then the DNA isolated using the Magnesil® KF, Genomic System on the Thermo Electron KingFisher® mL instrument. This extracted DNA was then tested by multiplex PCR for the Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2) genes. (3762)

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Brain Res. 1127, 66–75. Molecular characterization and gene expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the lizard brain. 2007

Valiante, S., Prisco, M., Capaldo, A., Zambrano, I., De Falco, M., Andreuccetti, P., Laforgia, V., and Varano, L.

Notes: The authors cloned pituitary adenylate cyclase-activating polypeptide (PACAP) from lizard (Podarcis sicula) brain. They then isolated total RNA from lizard brain using the SV Total RNA Isolation System and used 4µg of total RNA in a reverse transcription with ImProm-II™ Reverse Transcriptase and oligo(dT)15 primers at 37°C for 1.5 hours. The PACAP cDNA was amplified by PCR, and the resulting PCR products were cleaned up using the Wizard® SV Gel and PCR Clean-Up System prior to sequencing. (3666)

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Forensic Sci. Int. 48, 478–85. Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves. 2007

Davoren, J., Vanek, D., Konjhodzic, R., Crews, J., Huffine, E. and Parsons, T.J.

Notes: The authors compared two DNA extraction methods: the International Commission on Missing Persons silica method and the standard phenol:chloroform method to determine the preferred method for extraction of DNA from skeletal remains. The efficacy of DNA extraction was measured by real-time PCR to quantify DNA and to check for the presence of PCR inhibitors, and by amplification with the PowerPlex® 16 System. DNA was extracted from processed bone powder, and 10µl of the final extract was amplified using the PowerPlex® 16 System and GeneAmp® PCR System 9700 according to the manufacturer's recommendations, except that the extension time was doubled from 30 seconds to 60 seconds for the first 10 cycles and from 45 seconds to 90 seconds for the next 22 cycles. Amplified products were detected using the ABI PRISM® 3100 Genetic Analyzer. The authors concluded that the silica-based method gave better results in autosomal STR typing than the organic extraction method. (3818)

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J. Endocrinol. 197, 201–12. Neonatal exposure to bisphenol A modifies the abundance of estrogen receptor alpha transcripts with alternative 5'-untranslated regions in the female rat preoptic area. 2007

Monje, L., Varayoud, J., Luque, E.H. and Ramos, J.G.

Notes: The authors investigated the effect of neonatal bisphenol A (BPA) exposure in rats on expression of estrogen receptor α (ERα) transcripts. Alternative ERα transcripts in preoptic area of treated and untreated rats were quantified using real-time RT-PCR. Reverse transcription was performed using 4µg of total RNA, 200pmol random primers and 300 units M-MLV Reverse Transcriptase. Real-time PCR was performed using SYBR® Green I to quantify amplified products. To determine if the changes in BPA-induced ERα transcript expression were caused by DNA methylation, the methylation status of the five ERα promoters was examined by bisulfite modification. Genomic DNA was isolated from rat tissue using the Wizard® Genomic DNA Purification Kit, denatured with NaOH, then treated with hydroquinone and sodium bisulfite. Prior to methylation-specific PCR, DNA was cleaned up using the Wizard® DNA Purification Resin as directed by the manufacturer. PCR products were cleaned up again using the Wizard® SV Gel and PCR Clean-Up System, then subjected to restriction enzyme digestion and agarose gel electrophoresis to reveal methylation-dependent sequence differences. (3911)

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Infect. Immun. 75, 3478–89. Reversal of the antichlamydial activity of putative type III secretion inhibitors by iron. 2007

Slepenkin, A., Enquist, P.A., Hägglund, U., de la Maza, L.M., Elofsson, M. and Peterson, E.M.

Notes: The authors screened members of a class of acylated hydrazones of salicylaldehydes (INPs) to characterize their ability to inhibit growth of Chlamydia by affecting the type III secretion (T3S) system, a potent virulence mechanism. Expression levels of various T3S genes and gene markers of early, middle and late developmental cycles were examined by RT-PCR in Chlamydia trachomatis-infected HeLa 229 cells in the presence and absence of INPs. HeLa229 RNA was isolated at 4, 8, 24 and 36 hours postinfection, treated with RQ1 RNase-Free DNase and amplified using the Access RT-PCR System in the presence of 0.5 units of RNasin RNase Inhibitor. Each set of experiments included a no-reverse transcriptase control reaction to control for DNA contamination and a positive control reaction using the Positive Control RNA with Carrier and control primers supplied with the kit. (3795)

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Exp. Biol. Med. 232, 1195–1203. 13-cis-Retinoic acid alters intracellular serotonin, increases 5-HT1A receptor, and serotonin reuptake transporter levels in vitro. 2007

O'Reilly, K.C., Trent, S., Bailey, S.J. and Lane, M.A.

Notes: The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers. (3790)

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Cancer Res. 67, 10600–10607. Interaction of the tumor metastasis suppressor nonmetastatic protein 23 homologue H1 and estrogen receptor alpha alters estrogen-responsive gene expression. 2007

Curtis, C.D., Likhite, V.S., McLeod, I.X., Yates, J.R. and Nardulli, A.M.

Notes: Tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1) interacts with estrogen receptor α (ERα) and influences ERα-mediated gene expression. The authors knocked down NM23-H1 expression using RNA interference in estrogen-treated or untreated MC-7 human breast cancer cells and determined the effect on transcription of estrogen-responsive genes, including progesterone receptor, Bcl-2, cathepsin D and cyclin D1. Levels of these mRNAs were measured in the presence of NM23-H1 or control small interfering RNAs using quantitative RT-PCR. Total RNA was treated with RQ1 RNase-Free DNase to remove contaminating DNA, and cDNA was synthesized using the Reverse Transcription System. The resulting cDNA was subjected to quantitative PCR using SYBR® Green dye. (3789)

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J. Biol. Chem. 282, 21818–21828. Degradation of hsp70 and other mRNAs in Drosophila via the 5´ 3´ pathway and its regulation by heat shock. 2007

Bönisch, C., Temme, C., Moritz, B. and Wahle, E.

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-32P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq® DNA Polymerase, 1.5mM MgCl2 and 1X Green GoTaq® Flexi Reaction Buffer. (3707)

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Hum. Mutat. 0, 1-6. Novel Plexor SNP genotyping technology: comparisons with TaqMan and homogenous MassEXTEND MALDI-TOF mass spectrometry. 2007

Tindall, E.A., Speight, G., Petersen, D.C., Padilla, E.J., and Hayes, V.M.

Notes: This study compared the performance of the Plexor® qPCR System with the TaqMan® and MassEXTEND™ methods for genotyping analysis of 11 SNPs in >2000 DNA samples. All three methods were shown to be equivalent in call rate and accuracy. The Plexor® System is described as a cost-effective, efficient alternative to the TaqMan® technology for medium-throughput SNP analysis. Plexor® qPCR System reactions contained 5ng template DNA, 0.2µl 5µM allele-specific primers, 0.2µl 10µM anchor primer, and 2.5µl 2X Plexor™ Master Mix in a total volume of 5µl. PCR was performed on an ABI PRISM® 7900HT Sequence Detection System using the following cycling conditions: 95°C for 2 minutes; 50°C for 35s; and 40 cycles of 95°C for 5s, 60°C for 35s. Primers were designed using the Plexor® Primer Design Software. (3674)

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J. Forensic Sci. 52, 1073–6. Characterization of the variant allele 9.2 of Penta D locus. 2007

Miozzo, M.C., Maxzud, M.K., Pacharoni, C.M., Mutal, S.A. and Modesti, N.M.

Notes: During casework analysis using the PowerPlex® 16 System, the authors identified an off-ladder allele at the Penta D locus as the microvariant allele 9.2. DNA from individuals with the 9.2 allele was amplified using a single primer pair specific for Penta D, and the amplification products were purified and sequenced to characterize the microvariant allele. PCR products were purified using the Wizard® PCR Preps DNA Purification System. Sequence analysis revealed that the 9.2 allele has 10 STR repeats and a TAA deletion in the 3´ flanking region. (3771)

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J. Clin. Microbiol. 45, 1469–1477. Multilocus sequence typing of the pathogenic fungus Aspergillus fumigatus. 2007

Bain, J.M., Tavanti, A., Davidson, A.D., Jacobsen, M.D., Shaw, D., Gow, N.A. and Odds, F.C.

Notes: The authors developed a multilocus sequence typing scheme (MLST) to examine sequence variation and discriminate between Aspergillus fumigatus strains. They also examined the distribution of MAT1-1 and MAT1-2 sexual idiomorphs in 100 clinical and environmental isolates. Sexual idiomorphs were determined using PCR and a reverse primer to both idiomorphs and a forward primer specific to either MAT-1 or MAT-2. PCRs consisted of 2mM MgCl2, 200µM DNTPs and 2.5 units of GoTaq® DNA Polymerase. (3714)

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J. Biol. Chem. 282, 21798–21809. Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed. 2007

Panis, G., Méjean, V. and Ansaldi, M.

Notes: The authors studied the defective prophage KplE1 in E. coli K12 to map the binding sites of proteins required for recombination. Prior to in vivo excision assays in two E. coli K12 strains, the presence of three DNA sequences required for recombination was confirmed by PCR using GoTaq® DNA Polymerase. In vitro excision assays were also performed using linear and supercoiled DNA substrates that were purified using the Wizard® PCR Clean-Up System. Finally the phage-encoded integraseS (IntS) mRNA was quantitated by real-time RT-PCR. The RNA template was purified from E. coli K12 using the PureYield™ RNA Midiprep System. (3722)

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J. Mol. Endocrinol. 36, 449–461. Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo. 2007

Brown, K.A., Boerboom, D., Bouchard, N., Doré, M., Lussier, J.G. and Sirois, J.

Notes: The authors cloned the novel equine aldo-keto reductase AKR1C23 and characterized its expression patterns in the preovulatory follicle. The AKR1C23 cDNA was amplified from equine ovarian RNA using the Access RT-PCR System and primers designed by sequence alignments of known AKR sequences, then cloned into the pGEM®-T Easy Vector. Levels of AKR1C23 and ribosomal protein L17a mRNAs in various equine tissues were quantified using the Access RT-PCR System and 21 cycles and 18 cycles, respectively, followed by agarose gel electrophoresis, transfer to nylon membranes, and hybridization to radiolabeled probes synthesized using the Prime-a-Gene® Labeling System. (3791)

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J. Biol. Chem. 282, 29211–21. A novel CaV1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits. 2007

Cheng, X., Liu, J., Asuncion-Chin, M., Blaskova, E., Bannister, J.P., Dopico, A.M. and Jaggar, J.H.

Notes: The authors identified a novel subunit of the voltage-dependent L-type Ca2+ channel (CaV1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM®-T Easy Vector and sequenced using the T7 Promoter Primer. (3801)

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J. Immunol. 179, 4829–4839. Aging up-regulates expression of inflammatory mediators in mouse adipose tissue. 2007

Wu, D., Ren, Z., Pae, M., Guo, W., Cui, X., Merrill, A.H. and Meydani, S.N.

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

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Proc. Natl. Acad. Sci. USA 104, 10637–10642. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. 2007

Rendón, M.A., Saldaña, Z., Erdem, A.L., Monteiro-Neto, V., Vázquez, A., Kaper, J.B., Puente, J.L. and Girón, J.A.

Notes: The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector containing the ecpA gene, restored the strain's ability to adhere to epithelial cells. (3719)

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J. Clin. Invest. 117, 3042–3048. HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection. 2007

McAulay, K.A., Higgins, C.D., Macsween, K.F., Lake, A., Jarrett, R.F., Robertson, F.L., Williams, H. and Crawford, D.H.

Notes: The authors examined whether genetic differences at the HLA class I locus affect development of Epstein Barr Virus-associated diseases. Peripheral blood mononuclear cells were isolated from asymptomatic EBV-seropositive and seronegative individuals and patients with acute infectious mononucleosis. DNA was isolated, and genotypes at two HLA class I loci and one HLA class III locus, as a control, were determined by PCR. The 10µl PCRs contained 25ng of DNA, 1X GoTaq® Flexi Reaction Buffer, 2.5mM MgCl2, 200µM dNTP, 0.5 units of GoTaq® Flexi DNA Polymerase and 25µM of forward and reverse primer, one of which was labeled with 6-FAM fluorescent dye. The results show that HLA class I polymorphisms might predispose people to develop infectious mononucleosis upon EBV infection. (3712)

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Appl. Environ. Microbiol. 74, 811-817. High frequency of histamine-producing bacteria in enological environment and instability of the phenotype. 2007

Lucas, P.M., Claisse, O. and Lonvaud-Funel, A.

Notes: Because of concerns about the consumption of histamine in wine (which makes histamine more potent), the quantity of lactic acid bacteria (LAB), which can produce histamine during winemaking, was determined in 264 samples of red wine from 116 wineries. DNA was isolated from LAB strains grown in De Man Rogosa Sharpe (MRS) broth using the Wizard® Genomic DNA Purification Kit. Glass beads were used to disrupt the cells, and a modified Wizard® Genomic DNA Purification protocol that included PVP treatment was performed. The purified DNA was used in qPCR analysis. (3741)

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Am. J. Pathol. 171, 1153-1167. Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy. 2007

Bodó, E., Tobin, D.J., Kamenisch, Y., Bíró, T., Berneburg, M., Funk, W. and Paus, R.

Notes: To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan® Assay. (3746)

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Assay Drug Dev. Technol. 5, 237–245. A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP 2007

Kumar, M., Hsiao, K., Vidugiriene, J. and Goueli, S.A.

Notes: The authors of this paper introduce a luminescent assay to monitor changes in cellular cAMP concentration. The assay can be used to study the activity of G-protein coupled receptors that modulate adenylate cyclase activity. The assay is compatible with high-throughput screening in 96-, 384- and 1536-well formats. (3928)

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Mol. Cell. Biol. 26, 8448–8460. Specific isoforms of translation initiation factor 4GI show differences in translational activity. 2007

Coldwell, M.J. and Morley, S.J.

Notes: The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function. (3778)

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J. Virol. Methods 144, 86–90. A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus. 2007

Prichard, M.N., Daily, S.L., Jefferson, G.M., Perry, A.L. and Kern, E.R.

Notes: The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3761)

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